Immunoassays of s-adenosylmethionine and methylation index in personalized medicine and health evaluation

ABSTRACT

The invention provides a method of detecting the presence, absence or severity of a disease in a patient wherein said disease is accompanied by decreased level of S-adenosylmethionine, or increased level of S-adenosylhomocysterine, or reduced methylation index comprising: identifying any individual or a patient that is suspected of having said disease or is at risk of having said disease; obtaining a biological sample from said patient; determining the level of SAM in said biological sample using an antibody derived from a hapten analog of SAM, SAH; and correlating the levels of SAM, SAH and MI in said biological sample with the presence, absence, or severity of said disease. The invention also provides methods for determining methylation index in biological fluids which is indicative of the health status of an individual. Additionally, the invention includes colloidal gold test strips and homogenous enzyme immunoassays which are useful for determining S-adenosylmethionine and S-adenosylhomocysteine.

This application is a continuation-in-part of U.S. Ser. No. 14/457,099filed Aug. 11, 2014; which application is a continuation-in-part of U.S.Ser. No. 14/218,928 filed Mar. 18, 2014, the entire contents of whichare incorporated by reference herein. This application also claims thepriority benefit under 35 U.S.C. section 119 of U.S. Provisional PatentApplication No. 61/801,547 entitled “Immunoassay Of S-AdenosylmethionineIn Personalized Medicine And Health Or Cancer Evaluation” filed on Mar.15, 2013, and which is in its entirety herein incorporated by reference.

FIELD OF THE INVENTION

The present invention generally relates to measuring levels ofS-adenosylmethionine in biological fluids as a marker of many diseases.

The instant invention also relates to measuring levels ofS-adenosylmethionine in biological fluids as a marker of many diseasesby using antibodies raised against analogs thereof.

The present invention relates to measuring levels ofS-adenosylmethionine in biological fluids as a marker of disease andcorrelating the levels to disease progression and determining the propertherapeutic protocol based on the levels of S-adenosylmethionine.

This invention also relates to diagnostic, screening, and earlydetection methods for cancer, which can also be used to monitortherapeutic effectiveness and relapse monitoring in cancer and otherpathological and physiological processes.

The present invention is also directed to a system for developing targetspecific assays for determining whether a patient will likely respond toa target specific drug, and more particularly to a such a system that ishighly economical and provides synergies when diagnostics and drugs aredeveloped in parallel.

The instant invention is also directed to a method for discovering,screening, searching, identifying, developing and/or evaluating themeasurement of the methylation index for correlating disease progressionand disease treatments and response to said treatments.

The present invention also relates to using the methylation index (ormethylation status in some of the literature). In this filing we usemethylation index to represent SAM/SAH) as a biomarker, methods,devices, reagent, systems and kits for the detection, diagnosis ofcancer as well as other diseases and for the monitoring of cancerprogression and for monitoring the progress of various cancer treatmentsand other diseases. Cancer progression is characterized by progressivelyincreased levels of global DNA hypomethylation, regional CpGhypermethylation, and genomic instability. Decreased methylation indexis co-related with the global DNA hypomethylation and genomicinstability. Therefore, it is a good marker to help evaluate healthstatus and disease progression or stages.

BACKGROUND OF THE INVENTION

S-Adenosylmethionine (SAMe) is found in almost every tissue and fluid inthe body. SAM plays a crucial role in the process calledtransmethylation. Methylation is involved in nearly every aspect oflife. SAM is the primary “methyl” donor for a variety of methyl-transferreactions in DNA, RNA, proteins, lipids, and small molecules in thebody. Proper DNA methylation is essential for normal embryonicdevelopment. Methyl-transferase gene homozygously deleted (knocked out)has been proven lethal (Pegg, A. E., Feith, D. J., Fong, L. Y., Coleman,C. S., O'Brian, T. G., and Shantz, L. M., 2003, Biochem. Soc. Trans. 31,356-360). DNA improperly methylated has been found in many tumors.Alterations in DNA methylation patterns induce the expression ofoncogens or silence the expression of tumor suppressor genes, and methyldeficient diets have been shown to promote liver cancer in rodents.

The transsulfuration begins with S-adenosylhomocysteine (SAH), theresidual structure of SAM upon donating the methyl group(transmethylation). Hydrolysis of SAH yields homocysteine, which inturns converts to cystathionine, then cysteine, and eventually, toglutathione, the hepatocellular antioxidant and life-savingdetoxification agent.

The aminopropylation is another process initiated with SAM throughdecarboxylation. The decarboxylated SAM then couples with putrescine togenerate spermidine and spermine which are critical to cell growth,differentiation and the stability of DNA and RNA. Furthermore,Methylthioadenosine (MTA), the by-product of polyamine synthesis, is apowerful analgesic and anti-inflammatory agent. This may be, at leastpartially, responsible for the clinical benefits observed in thetreatment of osteoarthritis, rheumatoid arthritis and fibromyalgia withSAMe.

SAMe plays a role in the immune system, maintains cell membranes, andhelps produce and break down brain chemicals, such as serotonin,melatonin, and dopamine. Deficiency of either vitamin B12 or foliate canreduce the level of SAMe. SAMe is also an antioxidant, a substance thatprotects the body from damaging reactive oxygen molecules in the body.These reactive oxygen molecules can come from inside the body or fromenvironmental pollution and are thought to play a role in the agingprocess and the development of degenerative disease. In general, SAMe isthought to raise the level of functioning of other amino acids in thebody.

By way of further background, S-adenosyl-1-methionine is a substrate ofan enzyme lyase that converts S-adenosyl-1-methionine to the moleculemethylthioadenosine and homoserine; it is an aminobutyric chain donor totRNA; it is an aminoacidic chain donor in the biosynthesis of biotin;SAM-e, after decarboxylation, is the donor of aminopropyl groups for thebiosynthesis of neuroregulatory polyamines spermidine and spermine.(Zappia et al (1979), Biomedical and Pharmacologcial roles ofAdenosylmethionine and the Central Nervous System, page 1, PergamonPress. N.Y.)

SAM-e has been used clinically in the treatment of liver disease(Friedel H, Goa, K. L., and Benfield P., (1989),S-Adenosyl-1-methionine: a review of its pharmacological properties andtherapeutic potential in liver dysfunction and affective disorders inrelation to its physiological role in cell metabolism. Drugs. 38,389-416), arthritis (Di Padova C, (1987), S-adenosyl-1-methionine in thetreatment of osteoarthritis: review of the clinical studies. Am J. Med.83, (Suppl. 5), 6-65), and depression (Kagan, B, Sultzer D. L.,Rosenlicht N and Gerner R. (1990), Oral S-adenosylmethionine indepression: a randomized, double blind, placebo-controlled trial. Am. J.Psychiatry 147, 591-595.) Alzheimer's patients have reduced cerebralspinal fluid levels of S-adenosyl-1-methionine (Bottiglieri et al,(1990), Cerebrospinal fluid S-adenosyl-1-methionine in depression anddementia: effects of treatment with parenteral and oralS-adenosyl-1-methionine. J. Neurol. Neurosurg. Psychiatry 53,1096-1098.) In a preliminary study, SAM-e was able to produce cognitiveimprovement in patients with Alzheimer's disease. (Bottiglieri et al(1994), The clinical potential of admetionine (S-adenosyl-1-methioinine)in neurological disorders. Drugs 48, 137-152.) SAM-e brain levels inpatients with Alzheimer's disease are also severely decreased. (Morrisonet al, (1996), Brain S-adenosylmethionine levels are severely decreasedin Alzheimer's disease, Journal of Neurochemistry, 67, 1328-1331.)Patients with Parkinson's disease have also been shown to havesignificantly decreased blood levels of SAM-e. (Cheng et al, (1997),Levels of L-methionine S-adenosyltransferase activity in erythrocytesand concentrations of S-adenosylmethionine and S-adenosylhomocysteine inwhole blood of patients with Parkinson's disease. Experimental Neurology145, 580-585.)

SAM-e levels in patients treated with the antineoplastic drugmethotrexate are reduced. Neurotoxicity associated with this drug may beattenuated by co-administration of SAM-e. (Bottiglieri et al (1994), TheClinical Potential of Ademetionine (S-adenosylmethionine) inneurological disorders, Drugs, 48 (2), 137-152.)

Cerebral spinal fluid levels of SAM-e have been investigated in HIV AIDSdementia Complex/HIV encephalopathy and found to be significantly lowerthan in non-HIV infected patients. (Keating et al (1991), Evidence ofbrain methyltransferase inhibition and early brain involvement in HIVpositive patients Lancet: 337:935-9.) Additionally, it is also knownthat Pneumocystis Carinii pneumonia (PCP) occurs when the host isimmunosuppressed. The Pneumocystis pneumonia (PCP) in humans isassociated with advanced HIV disease, severe malnourishment in children,and treatments for cancers, advanced cancers, rheumatic disease, and theprevention of organ transplant rejection (Perez-Leal et al. Am J RespirCell Mol Biol Vol 45, PP1142-1146, 2011). It is fatal if untreated.Therefore early diagnosis is very important. Studies have been doneregarding S-adenosylmethionine (SAM) levels in the diagnosis ofPneumocystis Carinii Pneumonia (PCP) in patients with HIV Infection.Because S-adenosylmethionine is required by Pneumocystis carinii invitro, Pneumocystis infection depletes plasma SAM of rats and humans,nicotine reduces SAM of guinea pig lungs, and smoking correlates withreduced episodes of Pneumocystis pneumonia (PCP) in AIDS patients.Chronic nicotine treatment increases lung polyamine catabolic/anaboliccycling and/or excretion leading to increased SAM-consuming polyaminebiosynthesis and depletion of lung SAM (J. Biological Chemistry 2005;280(15):15219-15228). Therefore, severely decreased plasma SAM levelpredicts occurrence of PCP in patients with immunocompromised conditionsonly. The best treatment regimens for PCP should include keeping SAMlevel low as lowered SAM level helps to kill PCP pathogen, whereas,increasing SAM level is recommended for better outcomes of treatingother diseases when SAM or MI is low.

De La Cruz et al have shown that SAM-e, chronically administered, canmodify the oxidative status in the brain by enhancing anti-oxidativedefenses. (De La Cruz et al, (2000), Effects of chronic administrationof S-adenosyl-1-methionine on brain oxidative stress in rats.Naunyn-Schmiedeberg's Archives Pharmacol 361: 47-52.) This is similar toresults obtained with SAM-e in liver and kidney tissue. Thus SAM-e wouldbe useful as an antioxidant.

Oral SAM-e administration to patients with and without liver disease hasresulted in increases in liver glutathione levels. (Vendemiale G et al,(1989), Effect of oral S-adenosyl-1-methionine on hepatic glutathione inpatients with liver disease. Scand J Gastroenterol; 24: 407-15. Oraladministration of SAM-e to patients suffering from intrahepaticcholestasis had improvements in both the pruritus as well as thebiochemical markers of cholestasis. (Giudici et al, The use ofadmethionine (SAM-e) in the treatment of cholestatic liver disorders.Meta-analysis of clinical trials. In: Mato et al editors. MethionineMetabolism: Molecular Mechanism and Clinical Implications. Madrid: CSICPress; 1992 pp 67-79.) Oral SAM-e administration to patients sufferingfrom primary fibromyalgia resulted in significant improvement after ashort term trial. (Tavoni et al, Evaluation of S-adenosylmethioine inPrimary Fibromaylgia. The American Journal of Medicine, Vol 83 (suppl5A), pp 107-110, 1987.) SAM-e has been used for the treatment ofosteoarthritis as well. (Koenig B. A long-term (two years) clinicaltrial with S-adenosylmethionine for the treatment of osteoarthritis. TheAmerican Journal of Medicine, Vol 83 (suppl 5A), Nov. 20, 1987 pp 89-94)

SAM-e is clinically useful in many apparently unrelated areas because ofits important function in basic metabolic processes. One of its moststriking clinical uses is in the treatment of alcoholic liver cirrhosisthat, until now, remained medically untreatable. Mato et al demonstratedthe ability of oral SAM-e in alcoholic liver cirrhosis to decrease theoverall mortality and/or progression to liver transplant by 29% vs 12%as compared with a placebo treated group. (Mato et al (1999),S-adenosylmethionine in alcohol liver cirrhosis: a randomized,placebo-controlled, double blind, multi-center clinical trial, Journalof Hepatology, 30, 1081-1089.)

In alcoholic liver, SAM is reduced whereas SAH and Hcy levels areincreased. Two genes (MAT1A and MAT2A) encode for the essential enzymemethionine adenosyltransferase (MAT), which catalyzes the biosynthesisof S-adenosylmethionine (SAMe), the principal methyl donor and, in theliver, a precursor of glutathione. MAT1A is expressed mostly in theliver, whereas MAT2A is widely distributed. MAT2A is induced in theliver during periods of rapid growth and dedifferentiation. In humanhepatocellular carcinoma (HCC) MAT1A is replaced by MAT2A. This isimportant pathogenetically because MAT2A expression is associated withlower SAMe levels and faster growth, whereas exogenous SAMe treatmentinhibits growth (Lu, S C et al. Alcoho 35(3):227-34, 2005).

Sam-e also attenuates the damage caused by tumor necrosis factor alphaand can also decrease the amount of tumor necrosis factor alpha secretedby cells. Consequently, conditions in which this particular inflammatoryfactor is elevated would benefit from the administration of SAM-e.(Watson W H, Zhao Y, Chawla R K, (1999) Biochem J Aug. 15; 342 (Pt1):21-5. S-adenosylmethionine attenuates the lipopolysaccharide-inducedexpression of the gene for tumour necrosis factor alpha.) SAM-e has alsobeen studied for its ability to reduce the toxicity associated withadministration of cyclosporine A, a powerful immunosuppressor. (Galan A,et al, Cyclosporine A toxicity and effect of the s-adenosylmethionine,Ars Pharmaceutica, 40:3; 151-163, 1999.)

SAM-e, incubated in vitro with human erythrocytes, penetrates the cellmembrane and increases ATP within the cell thus restoring the cellshape. (Friedel et al, S-adenosyl-1-methionine: A review of itspharmacological properties and therapeutic potential in liverdysfunction and affective disorders in relation to its physiologicalrole in cell metabolism, Drugs 38 (3):389-416, 1989)

SAM-e has been studied in patients suffering from migraines and found tobe of benefit. (Friedel et al, S-adenosyl-1-methionine: A review of itspharmacological properties and therapeutic potential in liverdysfunction and affective disorders in relation to its physiologicalrole in cell metabolism, Drugs 38 (3): 389-416, 1989)

SAM-e has been administered to patients with peripheral occlusivearterial disease and was shown to reduce blood viscosity, chiefly viaits effect on erythrocyte deformability.

SAM-e is commercially available using fermentation technologies thatresult in SAM-e formulations varying between 60 and 80% purity. (Thatis, the final product contains 60-80% of the active or (S, S)-SAM-e and20-40% of the inactive or (R, S)-SAM-e.) (Gross, A., Geresh, S., andWhitesides, Gm (1983) Appl. Biochem. Biotech. 8, 415.) Enzymaticsynthetic methodologies have been reported to yield the inactive isomerin concentrations exceeding 60%. (Matos, J R, Rauschel F M, Wong, C H.S-Adenosylmethionine: Studies on Chemical and Enzymatic Synthesis.Biotechnology and Applied Biochemistry 9, 39-52 (1987). Enantiomericseparation technologies have been reported to resolve the pure activeenantiomer of SAM-e. (Matos, J R, Rauschel F M, Wong, C H.S-Adenosylmethionine: Studies on Chemical and Enzymatic Synthesis.Biotechnology and Applied Biochemistry 9, 39-52 (1987; Hoffman,Chromatographic Analysis of the Chiral and Covalent Instability ofS-adenosyl-1-methionine, Biochemistry 1986, 25 4444-4449: Segal D andEichler D, The Specificity of Interaction betweenS-adenosyl-1-methionine and a nucleolar 2-0-methyltransferase, Archivesof Biochemistry and Biophysics, Vol. 275, No. 2, December, pp. 334-343,1989) Newer separation technologies exist to resolve enantiomers on alarge commercial production scale at a very economic cost. In addition,it would be conceivable to synthesize the biologically active enantiomerusing special sterioselective methodologies but this has not beenaccomplished to date.

De la Haba first showed that the sulfur is chiral and that only one ofthe two possible configurations was synthesized and used biologically.(De la Haba et al J. Am. Chem. Soc. 81, 3975-3980, 1959) Methylation ofRNA and DNA is essential for normal cellular growth. This methylation iscarried out using SAM-e as the sole or major methyl donor with thereaction being carried out by a methyltransferase enzyme. Segal andEichler showed that the enzyme bound (S, S)-SAM-e 10 fold more tightlythan the biologically inactive (R, S)-SAM-e thus demonstrating a novelbinding stereospecificity at the sulfur chiral center. Othermethyltransferases have been reported to bind (R, S)-SAM-e to the sameextent as (S, S)-SAM-e and thus (R, S)-SAM-e could act as a competitiveinhibitor of that enzyme. (Segal D and Eichler D, The Specificity ofInteraction between S-adenosyl-1-methionine and a nucleolar2-0-methyltransferase, Archives of Biochemistry and Biophysics, Vol.275, No. 2, December pp. 334-343, 1989; Borchardt R T and Wu Y S,Potential inhibitors of S-adenosylmethionine-dependentmethyltransferases. Role of the Asymmetric Sulfonium Pole in theEnzymatic binding of S-adenosyl-1-methionine, Journal of MedicinalChemistry, 1976, Vol 19, No. 9, 1099-1103.)

SAM-e (whether in its optically pure enantiomeric form or in anenantiomeric or racemic mixture) presents certain difficult problems interms of its stability at ambient temperature that result in degradationof the molecule to undesirable degradation products. SAM-e (and thus itsenantiomers) must be further stabilized since it exhibits intramolecularinstability that causes the destabilization and breakdown of themolecule at both high as well as ambient temperatures. SAM-e hastherefore been the subject of many patents directed both towardsobtaining new stable salts, and towards the provision of preparationprocesses that can be implemented on an industrial scale. The presentpatent thus envisions the use of any of the salts of SAM-e alreadydisclosed in the prior art to stabilize the enantiomeric forms of SAM-e.

The clinical diagnostic field has seen a broad expansion in recentyears, both as to the variety of materials of interest that may bereadily and accurately determined, as well as the methods for thedetermination. Over the last several decades, testing for numeroussubstances such as drugs of abuse, or other biological molecules ofinterest has become commonplace. In recent years, immunoassay based onthe interaction of an antibody with an antigen has been extensivelyinvestigated for this purpose. Based on the unique specificity and highaffinity of antibodies, an immunoassay can accurately and preciselyquantitate substances at the very low concentrations found in biologicalfluids.

In view of the importance of SAM, it is desirable to have an easy andreliable method to measure its concentration in a biological sample. Aclassical assay method for measurement of SAM in rat liver utilized thetripolyphosphatase activity that was associated with5-adenosylmethionine synthetase in rat liver. The tripoly-phosphataseactivity is stimulated by low concentrations of S-adenosylmethionine.The assay sensitivity was reported at 0.1 nmole of SAM in an assayvolume of 0.1 ml (i.e. 10-6 M). Samples were lyophilized, homogenized inacid, and centrifuged. The supernatant was then passed through Dowex 1(HCO3-form) to remove endogenous inorganic phosphate and other potentialinterferons in the tissue. Great care has to be taken to avoid inorganicphosphate contamination from all reagents including the enzymepreparation, as well as glassware. The disadvantages of this assay areobviously lack of specificity, low sensitivity (1 hard to control andcompare between assays in different labs.

Another common method for measuring SAM in tissues or biological fluidsis HPLC or electrophoresis after sample preparation normallyencompassing the protein precipitation and/or extraction. Post columndetection may include derivatization, then measurement throughabsorption, fluorescence, or electrochemical change, and more recentlyby Mass Spectrometry (MS), or Tandem Mass Spectrometry (MS/MS) orLC-MS/MS were obtained. Radioisotopes or stable isotopic molecules ofSAM are frequently used for internal reference purpose. These methodsare capable of measuring low level of SAM in serum or plasma; however,the process typically is laborious, time consuming and/or requiresexpensive equipment. Another drawback is that it usually does notdistinguish the diastereoisomers of SAM at the sulfonium position. SAMis produced biologically in the (S,S) configuration at the sulfonium anda-aminoacid carbon respectively. Under normal physiological conditionsor storage conditions, SAM spontaneously racemizes to form a mixture of(R,S) and (S,S) isomers. Most methyltransferases are reported to bespecific to the (S,S) form of SAM only.

Another molecule of interest, S-Adenosylhomocysteine (SAH), is theprecursor leading to the biosynthesis of SAM, as well as the product ofall transmethylation reactions involving SAM as the methyl donor; i.e.,SAH is metabolically linked to SAM, and structurally it contains asingle carbon (as methyl) less than SAM. The co-existence and structuresimilarity of SAM and SAH present a great challenge to develop a methodfor the specific determination of the concentration of either moleculein a biological sample. The unstable (highly reactive) nature of SAMrenders the level of difficulty for its determination even higher.

As the immediate precursor of all of the homocysteine (HCys) produced inthe body, SAH has been suggested as a possibly more sensitive indicatorfor the risk of vascular disease than plasma HCys recently. The totalplasma concentration of SAH is normally much lower than HCys. Like SAM,with no distinguished absorption, the determination of SAH in serum orplasma has been a challenge. Advanced method such as LC with post columnderivatization, LC-MS/MS with internal reference is a recent developmentfor its determination. However, these methods typically involveexpensive instrumentations, laborious sample preparation, and timeconsuming procedures. Unlike SAM, however, SAH is a relatively stablecompound; the sample handling and stability are usually not a problem.

Since SAH is the product of all methylation reactions involving SAM asmethyl donor, increased concentration of SAH (or (SAH)) in tissues arefrequently accompanied by decreased concentration of SAM ((SAM)).Therefore, the ratio of (SAM) and (SAH) may be a more sensitiveindicator than the concentration of either SAM or SAH alone,particularly when their changes are subtle at early stages ofdysfunction or abnormal conditions. The ratio of the concentration ofSAM to the concentration of SAH known as “the methylation index” wasfirst proposed by Cantani, et al. as an indicator of the methylatingcapacity of the cell. The ratio was later referred by M. S. Hershfieldet al as methylation index (MI).

Therefore, a simple and convenient method that does not require costlyinstrumentation (LC, MS, and combinations) is clearly desirable for thedetermination of the biological concentration of SAM and to monitorchange and metabolic paths in the body fluids, tissues and organelles.With the monoclonal antibodies against SAM and SAH becoming available aspart of the instant invention, immunoassays will be available forresearch community and clinical labs to quantify SAM, SAH and MIconveniently, easily, accurately and quickly at low cost.

Similalrly and in view of the above, there is a need for improvedmethods for detection and diagnosis of cancer and other diseases, aswell as methods for monitoring the progress of the diseases andmonitoring the progress of various treatments for cancer and otherdiseases by quantitating the methylating index as a biomarker.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the improved synthetic process of the invention for makinghapten.

FIG. 2 shows the titers of two monoclonal antibody clones measured byELISA. The y-axis shows the OD₄₅₀ values. The x-axis shows the dilutionsof the purified ascites made at 1 μg/μl.

FIG. 3A shows the results of specificity of antibody clone #84-3. Crossreaction with analogs is less than 1%.

FIG. 3B shows the results of specificity of antibody clone #118-6. Crossreaction with analogs is less than 1%.

FIG. 4 shows the results of specificity of antibody clone #301-1. Crossreaction with analogs is less than 3%.

FIG. 5 shows the titer of HRP-anti-SAM (clone 118-6). The 0.25 μg/ml ofAdaM-PLL was coated on micro-titer wells. The HRP-anti-SAM was seriallydiluted and added to the wells. After incubation for about 60 minutes,substrates were added and OD450 was measured.

FIG. 6 Standard curve from direct competitive ELISA to quantify SAM.Lot#421212 is one batch of HRP-anti-SAM antibody 118-6 used. Differentdilution of HRP-anti-SAM (1:22000 to 1:32000) gave similar standardcurves.

FIG. 7 Standard curve from direct competitive ELISA to quantify SAH.HRP-anti-SAH antibody 301-3 was diluted at 1:2000 for the best result.

FIG. 8 illustrates the FCM results from normal liver cell line L02 andhepatocyte carcinoma cells line Hep G2 not stained with any antibody(FCM analysis control).

FIG. 9 illustrates the FCM results from normal liver cell line L02 andhepatocyte carcinoma cells line Hep G2 stained with anti-SAM monoclonalantibody from clone 84-3.

FIG. 10 shows the FCM results from normal liver cell line L02 andhepatocyte carcinoma cell line HepG2 stained with anti-SAM monoclonalantibody from clone 118-6.

FIG. 11 illustrates the FCM results from normal liver cell line L02 andhepatocyte carcinoma cell line Hep G2 stained with anti-SAM polyclonalantibody R3.

FIG. 12 show the FCM results from normal liver cell line L02 andhepatocyte carcinoma cell line stained with anti-SAH monoclonal antibodyfrom clone 301-1.

FIG. 13 shows the use of anti-SAM monoclonal antibody from clone 118-6in performing IHC with normal and cancerous breast pathological slide.

FIG. 14 illustrates use of anti-SAM monoclonal antibody from clone 118-6in performing IHC with normal and cancer lung pathological slides.

FIG. 15 shows the use of anti-SAM monoclonal antibody from clone 118-6in performing IHC with normal and cancerous liver pathological slides.

FIG. 16 illustrates the use of anti-SAM polyclonal antibody R3 inperforming IHC with normal and cancerous breast pathological slide.

FIG. 17 shows the use of anti-SAM monoclonal antibody from clone 118-6in performing IHC with normal and cancerous nephritic pathologicalslide.

FIG. 18 shows the use of anti-SAH monoclonal antibody from clone 301-1in IHC with normal and cancerous breast pathological slides.

FIG. 19A shows the SAM level for plasma samples from 4 normal volunteersstored at 4° C. over 10 days. The SAM level varied among individuals.

FIG. 19B describes the percentage of SAM degradation of plasma samplesfrom 4 normal volunteers stored at 5° C. over 10 days. SAM degradationvaried among individuals.

FIG. 19C illustrates the SAM level for plasma samples from 4 normalvolunteers stored at 15° C. over 4 days. The SAM level varied amongindividuals. SAM levels were reduced faster compared to the samplesstored at 4° C.

FIG. 19D shows the SAM and SAH levels at 15° C. over 10 days. Within 6hours, SAM was decreased while SAH was increased to more than 1000 nM.After about 3 days, both SAM and SAH started decreased fairly quickly.

FIG. 19E relates to the stability of SAM at 37° C. SAM was reducedquickly in all samples.

FIG. 19F illustrates the stability of SAM at 56° C. SAM was reduced evenfaster than at 37° C. in all samples.

FIG. 19G describes the stability of SAH at 56° C. SAH was first reducedquickly and then slightly increased around 1 hour, followed by furtherdecrease.

FIG. 20A illustrates that SAM level was significantly reduced afterdialysis with 20 mM phosphate buffer, pH 7.4. The curve for 37° C.dialysis for 24 hours showed the minimum detection value of 30 nM, whichshould be considered as trial or no SAM left.

FIG. 20B shows that SAH level was significantly after dialysis with 20mM phosphate buffer, pH 7.4. The curve for 37° C. dialysis for 24 hoursshowed the minimum detection value of 62.5 nM, which should beconsidered as trivial or no SAH left.

FIG. 21A relates to the stability of SAM at different pH. Low pH (acidicenvironment) helped prevent SAM from quick degradation. Basicenvironment speeded up SAM degradation.

FIG. 21B shows the stability of SAH at different pH. Both acid and baseincreased the speed SAH was degraded. Notice the SAH values at 1 daytime point could be higher than 1000 nM. The experiment only showed themaximum detection limit of 1000 nM, which caused the SAH spikes notshown for pH 7.0-7.5 and pH 5.5-6.0 curves.

FIG. 22A shows the means and distributions of SAM and SAH levels from 81normal serum samples. It fits a normal distribution (statisticalanalysis using R).

FIG. 22B shows the means and distributions of SAM and SAH levels from291 diseased samples. It fits a normal distribution (statisticalanalysis using R).

FIG. 23 describes normal SAM levels in different age groups anddifferent gender groups (Data from Study #2).

FIG. 24 illustrates normal SAM levels in different age groups anddifferent gender groups (data from Study #3). Women in all age groupswere detected with higher SAM levels in plasma than men. Average SAMlevels decrease with age.

SUMMARY OF THE INVENTION

The instant invention provides a method for providing cancer therapy ina mammal afflicted with cancer which method comprises the followingsteps: (a) determining the methylation index in a biological fluidsample of said mammal afflicted with cancer; (b) correlating saidmethylation index to disease progression in said mammal; and (c) basedon the results of (b) selecting the appropriate cancer therapeuticprotocol to treat said mammal afflicted with cancer.

The methylation index is measured by a method comprising the followingsteps: (a1) determining the concentration of S-adenosylmethionine insaid mammal wherein said method comprises: (i) obtaining a sample; (ii)mixing said sample with antibody specific for S-adenosylmethionine;(iii) detecting the binding of S-adenosylmethionine present in saidsample with said antibody; (iv) quantifying the binding as a measure ofthe amount of S-adenosylmethionine present in said sample; (a2)determining the concentration of S-adenosylhomocysteine according topublished literature procedures; and (a3) calculating the ratio of(a1)/(a2) to provide the methylation index of said biological sample.

The invention also provides a method for determining a cancer therapyregimen for treating a tumor in a patient comprising: (a) determiningthe methylation index in a patient sample; (b) comparing the level ofmethylation index obtained to a control methylation index to determinewhether the level of said index is a predictive marker; and b)determining a cancer therapy regimen for treating the tumor based on themethylation index values, wherein the methylation index values areindicative that the patient is either a responsive patient or anon-responsive patient.

The invention is also directed to a method for treating mood disordersin a human which method comprises: (a) determining the concentration ofS-adenosylmethionine in said human wherein said method comprises: (i)obtaining a sample; (ii) mixing said sample with antibody specific forS-adenosylmethionine; (iii) detecting the binding ofS-adenosylmethionine present in said sample with said antibody; (iv)quantifying the binding as a measure of the amount ofS-adenosylmethionine present in said sample; (b) correlating the levelsof SAMe with said mood disorders; and (c) based on the correlationresults of (b) administering effective amounts of a drug effective intreating said mood disorders.

The invention further provides a method for diagnosing in a subject, orpredicting the susceptibility of a subject to, a mental orneurodegenerative disorder, the method comprising: (a) obtaining one ormore biological samples from the subject; (b) determining the levels ofS-adenosylmethionine or the methylation index associated with saidsample; and (c) comparing the levels of the biomarkers determined in (b)with the levels of said biomarkers from one or more control samples,wherein abnormal levels of the two or more biomarkers in the sample(s)from the subject compared to the one or more control samples ispredictive of susceptibility of the subject to a mental orneurodegenerative disorder.

The invention also relates to a method of detecting the presence orabsence of a disease in a patient wherein said disease is accompanied bydeficient levels of S-adenosylmethionine comprising: identifying apatient that is suspected of having said disease or is at risk of havingsaid disease; obtaining a biological sample from said patient;determining the level of S-adenosylmethionine in said biological sampleusing an antibody derived from a hapten analog of S-adenosylmethionine;and correlating the level of S-adenosylmethionine in said biologicalsample with the presence or absence of said disease.

The invention is also directed to a method for assessing the need fortreatment of a subject with S-adenosylmethionine alone or in combinationwith other chemotherapeutic agents comprising the steps of: (a)collecting a sample of body fluid from a subject suspected of needingsuch treatment; (b) measuring the amount of S-adenosylmethionine levelsin said sample; (c) measuring the level of S-adenosylhomocysteine andcalculating the methylation index; (d) comparing the methylation indexof said sample with that of a normal standard; and (e) determining ifthe methylation index lies outside the normal range which is indicativeof a need for S-adenosylmethionine treatment.

The invention further provides a method for diagnosing or monitoring adisease or condition comprising the steps of: (a) obtaining a biologicalsample from a patient to be diagnosed or monitored; (b) determiningusing an immunoassay the quantity of SAM and SAH in said biologicalsample; (c) calculating the methylation index of said biological sample;and (d) determining if the quantity(ies) of said SAM, SAH andmethylation index in said biological sample is(are) indicative of thepresence, absence or status of the disease or condition.

The invention additionally provides a method of assessing one or morehealth statuses of a subject, the method comprising: determining themethylation index status in a test sample from the subject; saidmethylation index being calculated by determining the levels of SAM andSAH using any immunoassays; comparing one or more of the determinedmethylation index states to one or more baseline reference methylationindex states; wherein a difference, lack of a difference, or both in oneor more of the determined methylation index states and one or more ofthe baseline reference methylation index states indicates one or morestatuses of the subject.

The invention is also directed to a method of use comprising: (a)obtaining at least one sample from a subject; (b) determining the levelof SAM, SAH and methylation index as longevity predicting marker(s) inthe sample(s); and (c) predicting overall health of a subject based onthe marker level.

The invention further provides a method of monitoring an individual'shealth and relative risk for developing disease(s), comprising the stepsof: (a) collecting a sample from the individual; (b) measuring SAM andSAH levels in said sample using an immunoassay; calculating themethylation index; and therefore determining the individual's health.

The invention is also a method of use comprising: (a) treating a subjectwith a potential therapeutic intervention; (b) determining the effect ofthe treatment on the levels of SAM, SAH and methylation index as alongevity predicting marker; and (b) using the marker response topredict the effectiveness and prognosis of the intervention.

The invention also relates to a method of use comprising: (a) obtainingat least one sample from a subject; (b) determining the level of SAM,SAH and methylation index as longevity predicting markers in thesamples; (c) treating a subject with a potential therapeuticintervention based on the level of longevity predicting markers in thesubject; (d) determining the effect of the therapeutic intervention onthe level of a longevity predicting marker (only one marker?); and (e)using the marker response to predict the effectiveness and prognosis ofthe intervention.

The invention additionally provides a method for assessing patienthealth, the method comprising: providing a sample of bodily fluid from asubject; collecting the SAM and SAH content profile from the bodilyfluid; calculating the methylation index based on said SAM and SAHprofile, and comparing said methylation index profile to at least onereference methylation index profile to assess the health of the subject,the at least one reference methylation index profile profiling at leastone of: one or more disease, cerebrovascular diseases, Parkinson'sdisease, depression, diabetes, HBP, heart disease, inflammation, kidneydisease, liver diseases, pulmonary diseases, lung cancer, liver cancerand other cancers.

The invention further relates to a reference profile for assessingpatient health, the profile comprising levels of SAM, SAH andmethylation index that are differentially present at a level that isstatistically significant, the profile profiling being of at least oneof one or more disease, the at least one reference methylation indexprofile profiling at least one of: one or more disease, cerebrovasculardiseases, Parkinson's disease, depression, diabetes, HBP, heart disease,inflammation, kidney disease, liver diseases, pulmonary diseases, lungcancer, liver cancer and other cancers.

The invention also provides a method for assessing the cardiovascularhealth of a human comprising: (a) obtaining a biological sample from ahuman; (b) determining the levels of SAM, SAH and methylation index; (c)obtaining a dataset comprised of the levels of each of SAM, SAH andmethylation index; (d) inputting the data into an analyticalclassification process that uses the data to classify the biologicalsample, wherein the classification is selected from the group consistingof an atherosclerotic cardiovascular disease classification, a healthyclassification, a medication exposure classification, a no medicationexposure classification; and (e) determining a treatment regimen for thehuman based on the classification in step (d); wherein thecardiovascular health of the human is assessed.

The invention additionally provides a device for detecting and measuringthe presence of SAM, SAH and methylation index in a body fluid whereinthe device comprises a single unit for collecting the body fluid,analyzing the contents of the body fluid, and correlating the analysiswith physiological status.

The invention is also a method for determining the occurrence of adisease in a subject comprising the steps of: (a) providing a samplepreviously collected from said subject, (b) measuring at least onebiomarker in said sample, wherein the said biomarker is selected fromthe group of SAM, SAH and methylation index; and (c) determining theoccurrence of said disease from the biomarker values measured at step(b).

The invention further provides a method for diagnosing liver diseasecomprising: contacting an antibody which reacts withS-adenosylmethionine with a test sample to measure an amount of SAM; andrelating a measured amount of SAM in the test sample to a diagnosis ofwhether the test sample is from a person having the liver diseases.

The invention also provides a method for diagnosing liver diseasecomprising: contacting an antibody which reacts withS-adenosylmethionine with a test sample to measure an amount of SAM; andrelating a measured amount of SAM in the test sample to a diagnosis ofwhether the test sample is from a person having the liver diseases.

The invention further relates to a method for diagnosing a neurologicaldisease comprising: contacting an antibody which reacts withS-adenosylmethionine with a test sample to measure an amount of SAM,measuring an amount of SAM in the test sample; and relating a measuredamount of SAM in the test sample to a diagnosis of whether the testsample is from a person having the neurological disease.

The invention is also a method for diagnosing liver disease comprising:determining the methylation index (MI) of said sample and relating saidMI in the test sample to a diagnosis of whether the test sample is froma person having the liver disease.

The invention further includes determining the prognosis of acute andchronic liver disease in a subject by correlating the level of SAM inthe sample to the prognosis of said acute and chronic liver disease inthe subject.

The invention additionally provides a method for diagnosing neurologicaldisease comprising: determining the methylation index (MI) of saidsample and relating said MI in the test sample to a diagnosis of whetherthe test sample is from a person having the neurological disease.

The invention is also a multiplexed assay kit used to monitor liverhealth in a patient sample, said kit comprising an assay chamberconfigured to conduct a multiplexed assay measurement for: (a)methylation index level and another biomarker in said sample comprisingbilirubin (total or fractionated, conjugated or unconjugated), ammonia,carbohydrate-deficient transferring (CDT), alanine aminotransferase(ALT), alkaline phosphatase (ALP), serum glutamic pyruvic transaminase(SGPT), aspartate aminotransferase (AST), serum glutamic oxaloacetictransaminase (SGOT), albumin, total protein (i.e., plasma proteins),gamma-glutamyl transferase (GGT), gamma-glutamyl transpeptidase (GGTP),lactic acid dehydrogenase (LDH), prothrombin time, or combinationsthereof.

The invention also provides a method for monitoring liver health in apatient, said method comprising (a) obtaining a test sample from apatient; (b) measuring the methylation index level in said sample; (c)measuring the level of a biomarker selected from the group consisting ofbilirubin (total or fractionated, conjugated or unconjugated), ammonia,carbohydrate-deficient transferring (CDT), alanine aminotransferase(ALT), alkaline phosphatase (ALP), serum glutamic pyruvic transaminase(SGPT), aspartate aminotransferase (AST), serum glutamic oxaloacetictransaminase (SGOT), albumin, total protein (i.e., plasma proteins),gamma-glutamyl transferase (GGT), gamma-glutamyl transpeptidase (GGTP),lactic acid dehydrogenase (LDH), prothrombin time, or combinationsthereof; (d) comparing said methylation index level and said biomarkersin step (c) in said sample to a level of said methylation index and saidbiomarkers of step (c) in a normal control sample; and (e) diagnosingthe presence or absence of a liver disorder in said patient based onsaid comparison.

The invention also provides antibody which has substantially selectivereactivity with S-adenosylmethionine and has a cross reactivity of 10%or less with S-Adenosylhomocysteine, 10% or less with Adenosine, and 10%or less with L-Methionine.

The invention also provides antibody which has substantially selectivereactivity with S-adenosylmethionine and has a cross reactivity of 5% orless with S-Adenosylhomocysteine, 5% or less with Adenosine, and 5% orless with L-Methionine.

The invention also provides antibody which has substantially selectivereactivity with S-adenosylmethionine and has a cross reactivity of 1% orless with S-Adenosylhomocysteine, 1% or less with Adenosine, and 1% orless with L-Methionine.

The invention also provides antibody which has substantially selectivereactivity with S-adenosylmethionine and has a cross reactivity of 10%or less with S-Adenosylhomocysteine, 10% or less with Adenosine, and 10%or less with L-Methionine.

The present invention also provides monoclonal high affinity antibodiesimmunoreactive with S-adenosyl methionine wherein the binding affinityconstant of said antibodies for said S-adenosyl methionines is at least10⁶ M⁻¹.

The invention further provides an antibody which has substantiallyselective reactivity with S-adenosylmethionine and has a crossreactivity of 10% or less with S-Adenosylhomocysteine, 10% or less withAdenosine and 10% or less with L-Methionine and wherein said antibodyhas a binding affinity constant for said SAM or an analog thereof of atleast 10⁶M⁻¹.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides assays, diagnostics, therapeutics and medicalevaluation of patients with a variety of diseases where it is necessaryto assess their state of health. Their state of health can be assessedusing assays that provide accurate concentration of S-adenosylmethionineand S-adenosylhomocysteine. Having accurate determination of the abovemolecules will allow for calculation of the methylation index which isan important parameter related to the state of health of a human being.SAM measurement and methylation index may be an generic marker toevaluate healthy and diseased individuals.

The assay of the invention uses antibodies which are specific toS-adenosylmethionine and analogs thereof and prepared by inoculating ahost animal with an immunogen comprising an immunogenic substancedirectly or indirectly coupled to an S-adenosylmethionine hapten of theformula:

its enantiomers, diastereomers, enantiomerically enriched mixtures,racemic mixtures thereof, isotopically enriched forms thereof,crystalline forms, non-crystalline forms, amorphous forms thereof,charged and non-charged forms thereof, solvates thereof, metabolitesthereof, and salts thereof; wherein A is selected from the groupconsisting of

wherein M is selected from the group consisting of N, N⁺, C, S, S⁺, Se,Se⁺, and P; ----denotes the bonding location for each A group as definedabove;X is independently selected from the group consisting of H, CH₃, CH₂OH,CH₂NH₂, OH, OCH₃, NH₂, SH, CHO, and CN;Z is independently selected from the group consisting of CH₃, CH₂OH,CH₂NH₂, OH, OCH₃, NH₂, SH, CHO, and CN;B and C are independently selected from the group consisting of H, OH,NH₂, SH, F, Cl, Br, and I;D is independently selected from the group consisting of NH₂, OH, SH, F,Cl, Br, and I;Y is independently selected from the group consisting of H, CH₃, CH₂OH,CH₂NH₂, OH, OCH₃, NH₂, SH, CHO, and CN; andW is independently selected from the group consisting of H, COOH, CONH₂,COOCH₃, CN, CHO and functionalized derivatives thereof; and thereaftercollecting serum from said host animal. The antibodies are described indetail in commonly owned U.S. Pat. No. 8,344,115, the entire contents ofwhich are incorporated by reference as if it was denoted in itsentirety.

In another aspect, the invention provides mouse monoclonal and rabbitpolyclonal antibodies recombinant, humanized and chimeric antibodiesagainst S-adenosylmethionine and against S-adenosylhomocysteine usingthe analogs as described in commonly owned U.S. Pat. No. 8,344,115.

The present invention also provides monoclonal high affinity antibodiesimmunoreactive with S-adenosyl methionine wherein the binding affinityconstant of said antibodies for said S-adenosyl methionines is at least10⁶ M⁻¹ and also further provides an antibody which has substantiallyselective reactivity with S-adenosylmethionine and has a crossreactivity of 10% or less with S-Adenosylhomocysteine, 10% or less withAdenosine and 10% or less with L-Methionine and wherein said antibodyhas a binding affinity constant for said SAM or an analog thereof of atleast 10⁶M⁻¹.

As is well known in the literature, for the reaction: Ab+Ag=AbAg, therate of formation of the AbAg complex r_(f)=k_(forward)[Ab][Ag], and therate at which it breaks down r_(b) ⁼k_(backward)[AbAg] wherein[Ag]=Concentration of antigen; [Ab]=concentration of antibody;[AgAb]=concentration of AgAb complex. The rate constants k_(forward) andk_(backward) depends on temperature, pH and other conditions. Therefore,at equilibrium, r_(f)=r_(b).

The equilibrium or affinity constant Ka or K is defined from theequation:

$K = {\frac{k_{forward}}{k_{backward}} = \frac{\lbrack{AbAg}\rbrack}{\lbrack{Ab}\rbrack \lbrack{Ag}\rbrack}}$

Antibody-antigen interactions are in fact more complicated as both theantibody and antigen may have multiple binding sites. Avidity orfunctional affinity describes the extra tightness achieved by bindingthough multiple sites, which can be considerably greater than thesingle-site affinity. In practice, when we measure the binding of anantibody to an antigen, it is an average functional affinity which ismeasured and we do not worry about the exact number of binding sites.This functional affinity constant has an important bearing on theappropriate concentration of antibody needed for a particularapplication e.g. diagnostic assay or therapy.

Through the use of monoclonal hybridomally produced antibodies, thepresent inventors have now simultaneously improved the specificity andthe affinity of immunoassays, in all reactions between antiSAMantibodies and SAM. By increasing the affinity binding constant to thehigh values observed in the present invention, it is possible for thefirst time to carry out highly sensitive immunoassay procedures usingthe antibodies of the invention. The combined use of a hybridomallyproduced antibody and using a SAM analog allows the improvement inperformance.

The specificity relates to the lack of cross reactivity or lack ofinterference in the SAM-antibody reaction by other substances present inthe reaction mixture. Hybridomally produced antibodies in general,exhibit an extraordinary specificity since they are directed against asingle determinant. However because they are directed against a singledeterminant, they bind to fewer sites than classically (polyclonally)produced antibodies. This fact had been reflected in an existing fear inthe prior art that the extraordinary specificity of monoclonallyproduced antibodies, would be a detriment to their utilization insensitive clinically useful immunoassays. The present inventors howeverhave now demonstrated that hybridomally produced antibodies can detectless SAM than the conventionally produced antibodies, despite theirextraordinary specificity.

The antibodies of the invention have an affinity constant for saidS-adenosyl methionines of at least 10⁶ M⁻¹ and more preferably in therange of 10⁶ M⁻¹-10¹¹ M⁻¹.

The invention also provides immunoassays using monoclonal Anti-SAM andSAH antibodies to determine methylation index and the level of SAM indirecting and developing SAM treatment regimen and general healthevaluation, etc.

The invention further relates to using the methylation index as measuredin the present invention as a screen marker in the general state ofhealth of a given subject or population at large.

The antibodies of the invention are also useful in performingimmunohistochemical analysis ans flow cytometry analysis.

The instant invention further provides rapid, reliable and inexpensiveimmunoassays to measure SAM and SAH levels in saliva, urine, serum,plasma and whole blood semi-quantitatively using rapid test stripdevices. In an embodiment of the invention, a membrane is pre-soakedwith anti-SAM (or anti-SAH) antibody-dye (colloidal gold) conjugate.Secondary antibody is immobilized in the Control zone. Anti-SAM (oranti-SAH) antibody is immobilized in Test zone. Specimen migrates alongthe membrane. If SAM (or SAH) is present, antigen-antibody complex isformed and will be captured by antibodies in both Test and Controlzones, thus pink color is seen in both zones. If SAM (or SAH) is absent,antibody-dye conjugate is only captured by secondary antibody in theControl zone, thus pink band is seen only in control zone, whichindicate test has worked correctly and the results from the test linesshould be considered valid. Run standards and samples at the same timeand compare the signal (color and width of the positive band) strengthof test zones to those of standards to roughly determine theconcentration of SAM (or SAH), a way to semi-quantify SAM (or SAH).

In a competitive immunoassay similarly as above, Test zone isimmobilized with SAM (or SAH). The SAM (or SAH) from specimen competeswith the SAM (or SAH) immobilized on Test zone to the limited amount ofthe antibody-dye conjugates. The more SAM (or SAH) there is fromspecimen, the less pink line will be seen from Test zone. Extremely lowSAM (or SAH) or no SAM (or SAH) from specimen generates two strong linesin both Test and Control zones.

The semi-quantitative assay is ideal for consumers or patients to usebefore taking SAM-e as treatment for diseases, in the middle of SAM-etreatment, or to determine whether they should stop using SAM-e or not.

The invention also provides a method of personalized medicine for mammaldiseases, the method comprising measuring the methylation index in bodyfluids from a subject having a disease, and proposing a treatment with alikelihood of being effective for said subject based on the methylationindex levels in said body fluids.

The invention is also a method for monitoring the efficacy of a cancertreatment in a patient diagnosed with cancer comprising determining themethylation index level in the patient at a first point in time;treating the patient with a cancer treatment; determining themethylation index level in the patient at a second point in time; andcomparing the level(s) of the methylation index in the subject at thefirst point in time with the levels at the second point in time todetermine the efficacy of the cancer treatment.

In another aspect the invention provides a method for providing cancertherapy in a mammal afflicted with cancer which method comprises thefollowing steps: (a) determining the methylation index in a biologicalfluid sample of said mammal afflicted with cancer; (b) correlating saidmethylation index to disease progression in said mammal; and (c) basedon the results of (b) selecting the appropriate cancer therapeuticprotocol to treat said mammal afflicted with cancer. The method includescollecting blood samples from patients having stage I, or stage II, orstage III, or stage IV cancer and determining the levels of SAM and SAH,then calculating the methylation index, correlating the methylationindex with the cancer stage and then selecting an appropriatetherapeutic protocol for treating said mammal.

The invention is also useful in determining how well and effective DNAmethyltransferase inhibitors are in treating cancer. The methylationindex is the best tool or means to help evaluate how, the extent andspecificity of a certain DNA Methyltransferase (DNMT) inhibitors'functions in particular organs or tissues. Accordingly, the measurementof the methylation index can be used in assessing the effectiveness ofDNA methyl transferase inhibitors by using the measurements developed asa result of the present invention.

The invention is further directed to a method for predicting prognosisof a patient having a given disease, comprising: obtaining a tissuesample or a biological fluid sample from the patient; and measuring thelevels of SAM and SAH and calculating the MI in the sample, wherein themeasured levels, or a as compared to a reference levels, is indicativeof the prognosis of the disease is said patient.

The invention also provides an in vitro method for determining diseaseprognosis for a patient suffering from a given disease, said methodcomprising: (a) providing or obtaining a biological sample said patient;(b) measuring the amounts of SAM and SAH and calculating the MI of saidbiological sample; and (c) optionally deducing from the result of step(b) the prognosis of said patient.

The invention also provides a method of determining a subject'slikelihood of longevity which comprises comparing SAM levels from thesubject's plasma with the SAM levels from a control population, a highvalue of the subject's SAM level compared to the control populationindicating that the subject has an increased likelihood of longevity.

The instant invention also provides a method for determining whether animmunocompromised patient is susceptible to Pneumocystis Cariniipneumonia (PCP) infection which method comprises: (a) obtaining abiological sample from a patient; (b) determining using an immunoassaythe quantity of SAM in said biological sample; (c) comparing thequantity of SAM in said biological sample to the patient's baselinereference; and (d) determining if the quantity of said SAM in saidbiological sample is indicative of the presence, absence or status ofthe PCP infection.

The invention further provides:

1. Directed Therapies with SAMe

Both the effective studies on SAMe in treating mild to moderatedepression, osteoarthritis (better than nonsteroidal anti-inflammatorydrugs), fibromyalgia, and not so beneficial studies on SAMe have beenreported. The most possible reason for this is similar to most otherdiseases and treatments, i.e. certain patients are not good candidatesto use SAMe while some other patients are good candidates. To find outbeforehand whether patients are good candidates for using certainmedicine or not, some measurement has to be performed. Applicants' havediscovered that it is desirable to determine the level of SAM in bloodor urine samples before using SAMe for treatment of diseases.

Auxiliary treatment with SAMe in a variety of diseases, e.g. liverdisorders, B12 or foliate deficiencies, cancers, Parkinson's patientswho take Levodopa (L-dopa) has been accepted because these diseases cancause reduction of SAM level in the body. To be sure whether SAM levelis actually reduced, the best way is to directly measure the level ofSAM in blood plasma. There exist other situations when SAM level can bebrought down due to therapies and diseases themselves. Therefore,monitoring SAM level is very important in improving overall efficaciesof therapies whether the therapies include SAMe or not. For situationswhen SAM level is below certain acceptable level in the middle of othertreatment regimen for depression, osteoarthritis, fibromyalgia,Parkinson's, Alzheimer's disease, dementia, liver disorders, bursitis,tendonitis, chronic low back pain, multiple sclerosis, spinal cordinjuries, migraine headaches, lead poisoning, and to slow aging etc.,supplementing appropriate dosages of SAMe will benefit overalltreatment. For cases when treatment has not started, if SAM deficiencyis detected, administering SAMe via IV for the diseases above wouldquickly relieve the symptoms.

On the other hand, as the information on drug or food interactions withSAMe is very limited, plus the fact that SAMe is not without risk ofmore significant psychiatric and cardiovascular adverse effects,consumers should be instructed to avoid unmonitored consumption of thisdietary supplement until sufficient discussion has taken place withtheir primary healthcare provider (Fetrow, C. W. et al. “Efficacy of thedietary supplement 5-adenosyl-L-methionine.” Annals of Pharmacotherapy35 no. 11 (November 2001): 1414-1425). Taking SAMe with prescriptionanti-depressants can cause serotonin syndrome that can be quitedangerous. Immunoassay of SAM as describe in the U.S. Pat. No. 8,344,115is the best way to allow clinical labs and patients themselves toquickly find out the level of SAM. The immunoassays described in thepatent are sensitive, easy, quick, without using costly equipment. Theresults are comparable between assays. Furthermore, normal SAMconcentration in plasma appears to be different, greatly depending ongender (normally, men>women), individual's weight, and may be ethnicity,and diet, health condition, whether taking medicines or not, etc.Therefore, monitoring SAM level is critical in personalized and directedadministration of SAMe to achieve the best result in treatment.

2. Methylation Index in Disease Development and Prognosis

The methylation index is defined as a ratio of concentration of SAM toconcentration of SAH. It is important and more accurate to usemethylation index instead of the level of SAM itself under certaincircumstances. The reasons include (1) SAH+ is the direct end product ofSAM methylation reaction after methyltransferase (COMT). The SAHH isreversible enzyme whereas other enzymes are unidirectronal, theequilibrium dynamics of the SAHH reaction strongly favor SAH synthesisover homocystein synthesis (S J James, et al. Elevation ofS-Adenosylhomocysteine and DNA Hypomethylation: Potential EpigeneticMechanism for Homecysteine-Related Pathology. J. Nutri. 132:2361S-2366S,2002). The accumulation of SAH inhibits activities of methytransferases,thus, reduces the level of SAM. The moment SAM as the sole donor ofmethyl group in cells provides methyl group to DAN, RNA, Protein,phospholipids, neurotransmitters, peptides, hormones, etc., SAH isproduced. Therefore, the SAM/SAH is more sensitive and accurate inreflecting methylation reactions and an immediate and accurate indicatorof methylation status/level of the important molecules in living organsespecially when SAM fluctuation is subtle. (2) The level of SAM variesaccording to race, gender, body weight and diet, etc. Methylation indexcan reduce the variations caused by these and other factors.

Cancer is considered as both having genetics causes as well asepigenetic diseases. DNA methylation is one of the most importantepigenetic modifications. More and more findings are being revealed onthe importance of the once-neglected epigenetic influences on many lifephenomena, which says the impact of methylation on cancers could be moreand significant and in depth than what we know today. The level of DNAmethylation in cancer cells varies in different stages of cancerdevelopment. Abnormal DNA methylation occurs commonly in cancers in aspecial format of genome-wide hypo-methylation and regionalhyper-methylation. Global DNA hypo-methylation is associated withactivation of proto-oncogenes, such as c-JUN, c-MYC, and c-Ha-Ras, andgeneration of genomic instability. Hyper-methylation on CpG islandslocated in the promoter regions of tumor suppressor genes results intranscriptional silencing and genomic instability. CpG hyper-methylationacts as an alternative and/or complementary mechanism to gene mutationscausing gene inactivation, and it is now recognized as an importantmechanism in carcinogenesis. The inactivation of tumor-suppressor genes(e.g. p53 gene) by CpG-island hyper-methylation of the CpG islandslocated in their promoter regions is related to the cancer progressionand poor prognosis. Research results assign both therapeutic andchemo-preventive significance to methylation patterns in humanHepatocellular Carcinoma (HCC) and open the possibility of usingmolecular targets, including those identified in this study, toeffectively inhibit HCC development and progression (Diego F. Calvisiet 1. “Mechanistic and Prognostic Significance of Aberrant Methylationin the Molecular Pathogenesis of Human Hepatocellular Carcinoma.” J ClinInvest. 2007; 117(9):2713-2722.).

Drugs that are meant to reduce the level of methylation ofDNAs—demethylating agents, the promising chemotherapeutics drugs havebeen used and more are being studies to treat cancers (Esteller M. “DNAmethylation and cancer therapy: new developments and expectations.” CurrOpin Oncol. 2005 January; 17(1):55-60. 2005 January; 17(1):55-60.).

In the context of the present invention, “cancer” or “tumor” is intendedto include any neoplastic growth in a patient, including an initialtumor and any metastases. The cancer can be of the liquid or solid tumortype. Liquid tumors include tumors of hematological origin, including,e.g., myelomas (e.g., multiple myeloma), leukemias (e.g., Waldenstrom'ssyndrome, chronic lymphocytic leukemia, other leukemias), and lymphomas(e.g., B-cell lymphomas, non-Hodgkins lymphoma). Solid tumors canoriginate in organs, and include cancers such as lung, breast, prostate,ovary, colon, kidney, and liver. As used herein, cancer cells, includingtumor cells, refer to cells that divide at an abnormal (increased) rate.Cancer cells include, but are not limited to, carcinomas, such assquamous cell carcinoma, basal cell carcinoma, sweat gland carcinoma,sebaceous gland carcinoma, adenocarcinoma, papillary carcinoma,papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma,undifferentiated carcinoma, bronchogenic carcinoma, melanoma, renal cellcarcinoma, hepatoma-liver cell carcinoma, bile duct carcinoma,cholangiocarcinoma, papillary carcinoma, transitional cell carcinoma,choriocarcinoma, semonoma, embryonal carcinoma, mammary carcinomas,gastrointestinal carcinoma, colonic carcinomas, bladder carcinoma,prostate carcinoma, and squamous cell carcinoma of the neck and headregion; sarcomas, such as fibrosarcoma, myxosarcoma, liposarcoma,chondrosarcoma, osteogenic sarcoma, chordosarcoma, angiosarcoma,endotheliosarcoma, lymphangiosarcoma, synoviosarcoma andmesotheliosarcoma; hematologic cancers, such as myelomas, leukemias(e.g., acute myelogenous leukemia, chronic lymphocytic leukemia,granulocytic leukemia, monocytic leukemia, lymphocytic leukemia), andlymphomas (e.g., follicular lymphoma, mantle cell lymphoma, diffuselarge Bcell lymphoma, malignant lymphoma, plasmocytoma, reticulum cellsarcoma, or Hodgkins disease); and tumors of the nervous systemincluding glioma, meningoma, medulloblastoma, schwannoma or epidymoma.

3. Methylation Index in Embryo Development and Overall Human Health

The levels of SAM and SAH may be involved in the control of somaticembryogenesis by affecting the level of DNA methylation, which in turnmight cause differential changes in gene activation. An increase in thelevel of SAM may be a prerequisite for progression of embryogenesis andthe development of complete embryos (Munksgaard D, et al. “Somaticembryo development in carrot is associated with an increase in levels ofS-adenosylmethionine, S-adenosylhomocysteine and DNA methylation.”Physiologia Plantarum, Volume 93, Issue 1, Article first publishedonline: 9 Oct. 2008).

Methylation Index in General Human Health Evaluation and Screening

The levels of SAM and SAH may be involved in the control of somaticembryogenesis by affecting the level of DNA methylation, which in turnmight cause differential changes in gene activation. An increase in thelevel of SAM may be a prerequisite for progression of embryogenesis andthe development of complete embryos (Munksgaard D, et al. “Somaticembryo development in carrot is associated with an increase in levels ofS-adenosylmethionine, S-adenosylhomocysteine and DNA methylation.”Physiologia Plantarum, Volume 93, Issue 1, Article first publishedonline: 9 Oct. 2008).

Methyltransferase (MT) plays an important role in human diseasedevelopment. The Dopamine (DA) Hypothesis associated with the cause ofSchizophrenia describes an overactive DA pathway in Schizophreniapatients. Catechol-O-methyltransferase (COMT) degrades DA in centralnervous system. Inhibitors of COMT lay the foundation for treatment ofSchizophrenia (Renson J et al “Action of the inhibitors of catecholortho-methyl transferase on the adrenal catecholamines in the rat.” ArchInt Physiol Biochim. 1960 May; 68:534-7.). There are over 30 differentkinds of MT that work on various substances critical to the functions ofhuman being.

Methylation index is considered as an important indicator/marker forhuman general health, “vitality” indicators or “wellness” markers.

Furthermore, normal SAM concentration in plasma appears to be differentgreatly depending on gender (normally, men>women), individual's weight,and may be ethnicity/race, and diet, etc. Similarly dependency existsfor SAH concentrations since SAM and SAH are closely tied togethermetabolically. By utilizing the ratio of [SAM] and [SAH] it is likelythese variables can be eliminated or diminished.

Connection of SAM and SAH to cardiovascular disease, depression, cancerand aging-related diseases such as Alzheimer's disease is welldocumented. Methylation is highly critical in fetus development, indifferentiation, in epigenetic regulation of protein expression mainlyvia DNA, RNA and the histone methylation. The valuation of theS-adenosylmethionine and methylation capacity index is in theirscientific basis as “vitality” indicators or “wellness” markers.

In the present invention, non-invasive sample collection included urineand saliva sample collection. Measuring SAM and SAH levels from normalsubject were carried out with saliva sample as well. Oral micro biome isa complex ecological system where about 700 species of micr-oorganismsthat have been identified (Palmer Jr., et al. Community Development inBacterial Biofilms of the Oral Cavity. Microscopy and Microanalysis,2008). Some of the predominant groups present in the mouth includeStreptococcus, Neisseria and and other obligate anaerobes (Avila, M., etal. The Oral Microbiota: Living with a Permanent Guest. DNA & CellBiology, 2009). Oral organisms keep a mutual relationship with the hostby preventing pathogenic species from adhering to the mucosal surface.Some oral microflorae can cause dental plaques and are also a commoncause dental caries and periodontal disease. Oral disease in anindividual can be caused due to a combination of lack of oral hygieneand factors influencing the oral microbial community structure, such asdiet (Gibbons, R. J. et al. Bacterial Adherence in Oral Micr.obialEcology. Annual Review of Microbiology, 1975; Marsh, P. D. Role of theOral Microflora in Health Microbial Ecology in Health and Disease,2000). An understanding of the oral environment and microbialinteractions leads to understanding the main causes for oral diseases.Genetic factors determine metabolic profiles of each individual. It isanticipated that broad ranges or big standard deviations among samplesexist for many metabolites. The Examples 23-27 below indicated that bothsaliva and plasma samples have a relatively broad range of values. Thebest practice is to keep some factors such as diet, whether or nottaking certain medicines much similar between tests of SAM and SAH andcollect these samples at a fixed time of a day.

In the normal course of a physical examination, people have blood testswithout prior knowledge of diseases. They think they are disease free ornot sure whether they have any diseases or not. The MI indicator wouldgive them some idea of whether something has gone wrong or not withinthemselves, but not confirming what could be wrong. The lower the MI isfrom the individual's baseline reference MI, the stronger it is torecommend further testing or visiting doctors. It is proposed for thepurpose of health screening or disease prevention efforts.

The invention is of particular importance as it provides:

-   -   1. Direct, accurate and quantitative measurement of methylation        index with all types of bio-samples.    -   2. Direct, accurate and quantitative measurement of methylation        index in all types of lab settings.    -   3. Direct, accurate and quantitative measurement of methylation        index in relating to the evaluation of overall health        conditions; cancer prediction and prognosis; treatment (with or        without SAMe) evaluation of all diseases.    -   4. Direct, accurate and quantitative measurement of methylation        index in relating to differential diagnosis of cancers.    -   5. Direct, accurate and quantitative measurement of methylation        index in relating to chemotherapy resistance in cancer patients.    -   6. Direct, accurate and quantitative measurement of methylation        index in relating to the evaluation of fetal development,        differentiation and aging processes.    -   7. Semis-quantitative and qualitative immunoassay of methylation        index with stripes or other media for use conveniently and        easily by consumers in relating to reasons described in 1-6        above.    -   8. Semis-quantitative and qualitative immunoassay of SAM with        stripes or other media for use conveniently and easily by        consumers who take over-the-counter or prescribed SAMe for        various situations and diseases.    -   9. Semis-quantitative and qualitative immunoassay of SAM with        stripes or other media for use conveniently and easily by        consumers with urine and blood samples.    -   10. Quantitative assay of SAM in relating to the directed        medication of SAM-e for various reasons or purposes.    -   11. Direct, accurate and quantitative measurement of methylation        index with all types of bio-samples in relating to general        health evaluation.    -   12. Direct, accurate and quantitative measurement of methylation        index with all types of bio-samples in all types of lab        settings.    -   13. Direct, accurate and quantitative measurement of SAM level        with all types of bio-samples in assisting in determining or        adjusting SAMe treatment schemes.

As shown in Examples 23 and 24 of the present invention, there aresignificant differences in SAM content, SAH content and MI betweenhealthy people and people with disease, and accordingly the immunoassaysof the invention have substantial value in the clinical evaluation andhealth status of people. More in particular as shown in Table 16 ofExample 24, the summary shows the ranges and average of MI for theidentified cases. It is consistent with the t-test results that normalpeople has significantly higher MI (averagely 2.2) than diseasedpatients (average <1.56). The obvious difference between normal anddiseased group can also been seen from the maximum MI.

In the cases of cancers, we can see some samples had MI values fellbetween 4 and 6, therefore the average MI in cancer groups wererelatively higher than other diseased groups. The relatively higher MIin cancer samples is due to relatively higher SAM. This is consistentwith a report by Alissa K. et al (Chest. 2007. 132(4): 1247-1252.) inwhich serum SAM levels were elevated in patients with lung cancer ascompared to smokers with benign lung disorders and healthy nonsmokers.There were no significant correlations between SAM levels and tumor celltypes, nodule size, or other demographic variables. The temporarily highSAM levels may be caused by release of intracellular SAM from cells intoblood stream (as can be confirmed by IHC staining of SAM from cancercells) at some stage of cancer progression. Therefore, in our cancersamples, we could see wide ranges of MI values observed in all types ofcancers. As only certain stage of cancer development that SAM release issignificant, once release is completed, SAM level in blood stream won'tmaintain at the high level, which contributes to the big ranges of SAMlevels observed for all cancer types.

In the case of 68 cases of cerebrovascular diseases, there is only onesample has MI as 5.67. All others were less than 2.09. If we considerthat sample as an outlier, therefore remove it, the average MI for thatgroup would be less than 1. Therefore, cerebrovascular diseases would beamong other diseases in evaluating the range and average value of MI.

The invention also provides therapy with SAM-e and combination of SAM-ewith multiple therapeutic drugs. The invention is intended to includecompositions containing SAM-e and other therapeutic methods.

The present invention also provides an improved synthetic method formaking the Azaadenosyl(deamino) methionine hapten which is used to makeantibodies against SAM. The synthetic method is outlined in scheme 1below and in FIG. 1.

In carrying the method of the invention, blood samples are collectedfrom patients having a given disease condition and analyzed for SAM andSAH levels using ELISA methods and antibodies generated according to themethod of the invention. The invention may use enzyme labeledantibodies, but other labeling means such as a fluorescence substance,radioactive materials and biotin are also within the scope of theinvention. The ELISA Assay Format is as follows:

Format 1:

Sample or calibrator(s), (2) Antibody, (3) Hapten-Enzyme Conjugate, (4)Secondary antibody coated strips/microtiter plates, (Examples ofsecondary antibody: goat-anti-mouse antibody or goat-anti-rabbitantibody) (5) Wash solution, (6) Substrate(s). (7) Stopping reagent(optional if “end point” mode is used; for “rate” mode there is no needof a stopping reagent.) Format 2:

(1) Sample or calibrator(s), (2) Antibody, (3) Secondary antibody-EnzymeConjugate, (4) Immunogen (Hapten-carrier protein) coatedstrips/microtiter plates, (5) Wash solution, (6) Substrate(s and, (7)Stopping reagent (optional if end point mode is used; for “rate” modethere is no need of a stopping reagent.

Format 3:

(1) Two paired antibodies against two different epitopes of a molecule,(2) Sample or calibrator(s), (3) One antibody in (1) is conjugated withenzymes. (4) Wash solution, (5) Substrate(s). (6) Stopping reagent(optional if “end point” mode is used; for “rate” mode there is no needof a stopping reagent.)

The present invention also provides test kits which are based on animmunoassay (e.g., the ELISA test) for the immunological detection ofSAM which contain in addition to antibody against S-adenosylmethionine.The ELISA test kits can be in the any of the ELISA formats above. Forexample, the following components:(a) secondary Ab attached to solidphase; (b) immobilized hapten, hapten derivative, immunogen or alike;(c) enzyme substrate(s) in solid or dissolved form; (d) labeled haptenor derivatives (tracer or enzyme conjugates); (e) buffering and washingsolutions; (f) additives to prevent, for example, nonspecific adsorptionand aggregation; and (g) pipettes, incubation vessels, referencestandards, calibration curves, and color tables.

Once the levels of SAM and SAH are determined, the methylation indecesare calculated and used to determine the state of health of theindividual.

Generally speaking the average levels of SAM in healthy individuals wasabout 147±16 nM, the SAH level was 29±11 nM based on measurements from11 healthy individuals. The methylation index was 5±1. The normalmethylation index is above 4.

The average level of SAM for cancer patients was 103±52 nM.

The average level of SAM was 113±15 nM on patients with atherosclerosis.Preliminary results from SAH quantitative ELISA using rabbit monoclonalantibody against SAH showed substantially higher levels for SAHtherefore, the SAM/SAH is reduced significantly in patients withArteriosclerosis.

The average level of SAM in plasma is 45±8 nM (from 26 samples) forpatients with liver disorders and therefore much lower than that ofnormal people.

The ratio of SAM and SAH level is calculated and called methylationindex, which is a more accurate and convincing measure to evaluategeneral health, disease status, development and prognostics than asingle value of either SAM or SAH. Normally the methylation index is >4.In some pathological situations, it is less than 4 or even less than 1due to decreased SAM level and increased SAH level. The reducedmethylation index in turn will affect the methylation processes of manyimportant molecules such as DNA, RNA, peptides, hormone,neurotransmitters, etc.

The methylation index is used to determine a chemotherapeutic protocol.Cancer patients with significantly reduced methylation index levels aretreated with more aggressive protocols. The methylation index iscorrelated with the stage of the cancer to select an appropriate therapyfor each patient.

In another aspect of the invention, We are able to produce a sensitivitydetecting cutoff for SAM around 50 nM with the test strip method. Thecutoff value for SAH strip is around 200-500 nM. The sensitivity itselfis not essential compared to clinically meaningful cutoff values forparticular clinical purposes, yet having the ability to obtain highersensitivity is always good and have other uses.

Rapid test strips are made in the form of cassette and stick for testingSAM and SAH simultaneously or individually. When the product is madewith SAM strip alone, it is used for quickly testing SAM level in bloodand urine to be used as a way to direct SAM treatment, assist in diseasediagnostics and prognostics, and very likely act as a general healthindicator. When the product is made with SAH strip alone, it is used inevaluating status of diseases when hyperhomocysteinemia orhyper-S-adenosylhomocysteinemia is considered as a concern or issuspected of. When assembling both SAM and SAH strips in one unit,methylation index strip is made. When the cutoff of methylation index isestablished, qualitatively measuring the methylation index is anaccurate and better way of interpreting perturbation and problems ofmethionine cycle in human body and other organisms.

EXAMPLES

The following examples are intended to demonstrate the usefulness of themethods and therapeutic compositions of the present invention and shouldnot be construed to limit the scope of the invention in anyway. In thepresent specification the term biological sample is intended to includesaliva, urine, blood, serum, plasma, brain fluids, cerebrospinal fluids,tissue samples and cells or anything derived from the body of a mammalincluding a human.

Example 1 Generation of Monoclonal and Polyclonal Antibodies Against SAMand SAH Reagents:

-   AdaM: Azaadenosyl(deamino)methionine-   ASAM: Aza-SAM, or Nitrogen (N)-adenosylmethionine-   BgG: Bovine gamma globulinBSA: Bovine serum albumin-   BTG: Bovine thyrogloblulinCSAM: Carbon (C)-adenosylmethionine or    6(s)-Methyl-6-deaminosinefungindaH:    Deamino-5-adenosylhomocysteinedaHSO: daH sulfoxide DCC:    N,N′-dicyclohexylcarbodiimide-   DMF: Dimethylformamide-   EDAC: 1-Ethyl 3-(3-Dimethylaminopropyl)carbodiimide-   ELISA: enzyme-linked immunosorbant assay GAM plate/strip:    goat-anti-mouse IgG coated microplate or stripGAR plate/strip:    goat-anti-rabbit IgG coated microplate or stripHRP: horse radish    peroxidaseIB: Incubation bufferKLH: Keho lympet hemocyanine-   NHS: N-Hydroxysuccinamide-   PBS: phosphate-buffered saline-   RT: retention time (for HPLC) or room temperature-   SAH: S-Adenosylhomocysteine SAM: S-Adenosylmethionine-   KLH: Keyhole Limpet Hemocyanin-   EDC: 1-Ethyl-3-(3-Dimethylaminopropyl) Carbodiimide-   BSA: Bovine Serum Albumin-   PLL: Poly-lysine-   HRP: Horse Radish Peroxidase

1. Preparation of AdaM-NHS: To a flask containing overnight vacuum-diedAdaM (15.1 mg, ca. 0.041 mmole) was added 21.7 mg (0.107 mmole) of DCCand 7.2 mg (0.061 mmole) NHS. The solid mass was left on vacuum line for3-4 hr drying. Approximately 1.5 mL dry DMF was then added to the flaskunder nitrogen atmosphere, and then seal the flask. The solution wasstirred at RT overnight. TLC (10% MeOH in CH2Cl2) analysis indicated theformation of the NHS ester.

2. Preparation of AdaM-BSA: Weighed out 59.8 mg BSA to a round bottomedflask and added 5 ml freshly prepared 100 mM sodium phosphate solution,pH 8.25. Place the BSA solution in a 4° C. water bath with vigorousstirring. The AdaM-NHS prepared as described above was then slowly addedin 10 μl aliquot every few minutes. After a total of 150 μl was added,the conjugation mixture became turning cloudy. One milliliter of DMSOsolution was added to aid dissolution. Upon addition of another 50 μlAdaM-NHS in DMF, the mixture turned cloudy again. Water bath sonicationwas then applied for 5 minutes after every 10 μl×5 of AdaM-NHS wasadded. At the conclusion of 150 μl in total of AdaM-NHS in DMF wasadded, the mixture was sonicated for 20 minutes. To insure the conjugatewas free from any hapten, the pool was dialysis against PBS (1.5liter×4) over 2 days. The final volume of the conjugate is approximately36 ml, at estimated 1.66 mg/ml BSA.

3. Preparation of AdaM-KLH: Using the method above, weighed out 17.5 mgKLH, AdaM 15.1 mg. The final volume after dialysis is 29.5 ml withconcentration of 0.6 mg/ml.

4. Preparation of AdaM-PLL: AdaM 4.72 mg was dissolved in 1 ml DMF,EDC.HC16.5 mg and NHS 4.0 mg were added, then the mixture waswell-sealed, stirred at room temperature in dark overnight. Weighed out1.5 mg PLL dissolved with 1 ml 10 mM PBS pH 8.2. The activated AdaM wasthen added slowly to the PLL solution and the mixture was left overnightin dark. Dialyzed the reaction mixture for 48 hours with 10 mM PBS pH7.3. The final volume after dialysis is 3.5 ml with concentration of 1.4mg/ml.

5. Preparation of SAH-BSA: SAH (Sigma) 3.8 mg was dissolved in 1.5 mlPBS, EDC.HCl 10 mg and NHS 4.5 mg were added, the mixture waswell-sealed, stirred at room temperature in dark for 24 hours. Weighedout 12.9 mg BSA dissolved with 2 ml 10 mM PBS pH 7.8. The SAH solutionwas added slowly to the BSA solution and the mixture was left at 4° C.in dark overnight with stir. Dialyzed the reaction mixture for 72 hourswith 10 mM PBS pH 7.3. The final volume after dialysis is 8.4 ml withconcentration of 1.4 mg/ml.

6. Preparation of SAH-PLL: SAH 1.5 mg was dissolved in 1 ml PBS, EDC.HCl4 mg and NHS 2 mg were added, then the mixture was well-sealed, stirredat room temperature in dark overnight. Weighed out 1.5 mg PLL dissolvedwith 4.7 ml 50 mM PBS pH 9.6. The activated SAH was then added slowly tothe PLL solution and the mixture was left overnight in dark. Dialyzedthe reaction mixture for 48 hours with 10 mM PBS pH 7.3. The finalvolume after dialysis is 7.0 ml with concentration of 0.93 mg/ml.

Preparation of AdaM-HRP: The procedure for HRP conjugation is similar tothat of AdaM-BSA. Weight out 13.8 mg HRP powder (Sigma-Aldrich.) anddissolve it in 2 ml 100 mM sodium bicarbonate buffer, pH. 8.96, in around bottomed flask. 10 ul aliquot of AdaM-NHS in DMF (10 mg AdaM, EDC28.2 mg, NHS 10 mg were dissolved in 2 ml DMF, stirred mix for at least30 minutes) was then added slowly with stir. After about 40 minutes, twosolutions were mixed, and further dialyzed with PBS (1.5 liter×4) over2-3 days.

7. General Procedure for Generating Monoclonal Antibodies Against SAMand SAH:

Mouse monoclonal production is a common practice, based on the proceduredeveloped by the pioneer work of Kolher and Milstein (Nature, 256,495-497, 1975). Balb/c mice were used for monoclonal antibodyimmunization and ascites production. Immunization (1 ml total volume)was carried out with subcutaneous injections at multiple sites. Initialinjection utilizes 1:1 mixture of complete Freund Adjuvant and AdaM-BSAas well as AdaM-KLH conjugate solutions in PBS upon emulsification.Subsequent injections use incomplete Freund adjuvant.

Blood was collected periodically from immunized animals and cells wereremoved by centrifugation. Antisera thus obtained were then evaluated todetermine the immune response and the antibody titer. Depending onapplication, antibody may be used directly. When necessary, they can befurther purified to immunoglobulin level with ammonium sulfate or sodiumsulfate or by protein A column chromatography, etc.

For monoclonal antibody, once the clone is obtained it can be injectedinto host for ascites production. Antibody was then purified from theascites fluids by protein A affinity column. The hybridoma clone canalso be cultured on hollow fiber method to produce antibody.

Mice were primed with intravenous injection of immunogen three daysprior to its termination. The spleen of the mouse was harvested andhomogenized with a French Press. The spleen cells were then fused withmyeloma NS-1 cells in 5:1 ratio. The fused cell suspension was thenplated out on 96 wells microtiter plates. The hybridoma cell lines weregrown on RPMI1640 enriched with 18% fetal bovine serum, HAT and HTsupplements and screened. Clones that are positives to AdaM-PLLconjugate were selected for further studies. Final selection was basedon assay performance and cross activity profile. Selected clones werethen injected into mice to produce ascites fluid.

Through serial screening and selection, we identified a few clones thathave a better specificity and less cross reaction with other analogs.The titers of two monoclonal antibodies were tested and the results areshown in FIG. 2.

8. Rabbit Polyclonal Antibodies Against SAM and SAH

New Zealand White rabbits were used for polyclonal antibody production.Immunization (1 ml total volume) was carried out with subcutaneousinjections at multiple sites. The immunization process is the same aswhen immunizing mouse for monoclonal production. The rabbit antiserumwas test and the titer was above 1:12000 for both anti-SAM and anti-SAHseria before seria were collected.

Regarding titer of monoclonal antibody, the concentration of themonoclonal antibody is adjusted to 1 mg/ml. Using Immunoassay with HRP,we have got different titers depending on the amount of antigen used andat what OD level the best condition is considered. The titers in therange of 1:1000 to 1:500,000 have been tested.

In our experiments, hundreds of thousands of hybridoma cell lines wereobtained, each was tested for its specificity to SAM, SAH, Ade, Met.Binding of the monoclonal antibodies to SAM was much favored over otherthree analogs mentioned above. Using competitive ELISA, cross reactionto the analogs were analyzed in reference to reaction to SAM. Crossreaction to SAH at 10% and below were tested. FIG. 3A and FIG. 3B showthe two monoclonal antibody ones 84-3 and 118-6 with the lowest crossreaction to SAH.

Example 2 Antibody Specificity

To further test the specificity of the clones, cross reactions of clone#84 (similar results on #84 sub-clones) with all tested analogsincluding with SAH, methionine and with adenosine are very low, all <1%(could go further lower) (See FIG. 3A). Three analogs used in the crossreaction are SAH, methionine and adenosine. About 100 folds higherdosages of analogs than that of SAM was used in competitive ELISA. At 10μM dosage of the SAH, methionine and adenosine, competition of coatedantigen did not occur. However, no inhibition was seen by three analogs,the inhibition was clearly seen when SAM was added at a much lowerdosage than those of the analogs.

Cross reactions of clone #118-6 (similar results with other #118 subclones) with all tested analogs including with SAH, methionine and withadenosine are very low, all <1% (could go further lower) (FIG. 3B). Theresults were similar as clone #84-3 (similar results with other #84-3sub-clones). FIG. 4 showed the specificity characterization data formouse anti-SAH antibody clone #301 (similar for any #301 sub-clones).The calculated cross reaction to SAM is less than 3%, to other testedanalogs including with Homocysteine (H-Cys), L-Cysteine (L-Cys),Adenosine (Ade), Glutathione (GST), L-Cystathionine (L-CTT) are verylow, all <1% (could go further lower).

Example 3 Antibody Sensitivity

The sensitivity, the lowest detectable value in sample that is definedby the antigen concentration corresponding to the OD450 values that iscalculated by adding OD 450 value of blank wells plus 2 or 3 folds ofthe standard deviation, is about 3 nM (data not shown). Table 1A andTable 1B showed sensitivity experiment using clone #118-6 and #84-3,respectively. Table 1A shows cELISA result with 0.05 PLL-aza-SAM coatedmicro-titer plate, HRP-anti-118-6 at dilution of 1:10,000 in PBSincubation system. Different amounts of coating antigen, antibody, andincubating buffer will give slightly different minimum detection limits.The data below showed the minimum detection limit, which was calculatedby OD (when antigen=0)−2×Standard deviation=0.65205−2×0.072761=0.5065,was about 7.8125 nM. The results from Table 1B was calculated by OD(when antigen=0)−2×Standard deviation=1.00275−2×0.01223=0.97829, wasbetween 7.8125 to 15.625 nM. With other detecting technology, e.g.chemiluminescent assay and radioactive labeling technology, sensitivitymay be further increased.

TABLE 1A SAM Standard curve (#118-6) Standard (nM) OD 450 OD450 0 0.60060.7035 3.90625 0.6390 0.6405 7.8125 0.4320 0.5738 15.625 0.4033 0.406331.25 0.2752 0.3879 62.5 0.2039 0.2123 125 0.1547 0.1561 250 0.08660.1012

TABLE 1B SAM Standard curve (#84-3) Standards (nM) OD450 OD450 0 0.99271.0128 3.90625 0.8498 0.9258 7.8125 0.8769 0.9386 15.625 1.0222 1.026531.25 0.6903 0.5367 62.5 0.3895 0.3915 125 0.5111 0.4804 250 0.31060.2383

TABLE 1C SAH Standard curve SAH(nM) OD450 OD450 OD450 Mean Stdev 2500.4116 0.4426 0.442 0.4321 0.0177 125 0.5421 0.6358 0.5372 0.5717 0.055562.5 0.6055 0.7115 0.6444 0.6538 0.0536 31.25 0.7285 0.8199 0.75450.7676 0.0471 15.625 0.8045 0.8273 0.7975 0.8098 0.0156 7.8125 0.76020.8113 0.7708 0.7808 0.0270 3.906 0.7722 0.9144 0.7786 0.8217 0.0803 00.877 0.8992 0.8329 0.8697 0.0337

The cELISA result from Table 1C with obtained from 0.5 μg/ml BSA-SAHcoated micro-titer plate, HRP-anti-301-1 at dilution of 1:1000 in PBSincubation system. Different amounts of coating antigen, antibody, andincubating buffer will give slightly different minimum detection limits.The data showed the minimum detection limit, which was calculated by OD(when SAH Na=0)−2×Standard deviation=0.8697−2×0.033747=0.8022, wasaround 15.625 nM.

The titers were tested and were in the range of 1:1000 to 1:500,000 forthe mouse anti-SAM antibodies and 1:4000-8000 for mouse anti-SAHantibody with purified antibodies at 1 mg/ml. We have got differenttiters depending on the amount of antigen used and at what OD level thebest condition is judged, etc.

Example 4 Antibody Affinity

To measure the affinity, high (0.1 μg/ml) and low (0.05 μg/ml)concentrations of PLL-aza-SAM (AdaM-PLL) to coat 96-well micro-plate(Costa polystyrene flat bottom 96-well high bind stripwell clearmicroplate) were used, antibodies are diluted as shown in Table 2 from 1mg/ml stock concentration. The OD450 were read after HRP-anti-mouse IgGor HRP-anti-rabbit IgG and TMB substrates were added.

TABLE 2A Anti-SAM antibodies (118-6) binding to different amount ofantigen Monoclonal anti-SAM 118-6 PLL-aza-SAM Dilution 0.1 μg/ml 0.2μg/ml 100 4.4974 4.59425 200 4.2144 4.71145 400 4.0508 4.82100 8003.9753 4.89140 1600 3.8187 4.86745 3200 3.6592 4.78590 6400 3.33004.77320 12800 3.2063 4.24435 25600 2.6556 3.60425 51200 1.8867 2.44935102400 1.2460 1.60970 204800 0.7552 0.98320 409600 0.4396 0.54150 8192000.2514 0.28610

From Table 2A, for 118-6 antibody, when coating antigen was at 0.2μg/ml, half the maximum OD was seen at about 1:54800. The correspondingantibody concentration was [Ab]=(1 mg/ml/160,000g/mol)/54800=1.14×10⁻¹⁰, where 160,000 is the molecular weight ofantibody. When coating antigen was at 0.1 μg/ml, half the maximum OD wasobserved at about 1:44300. The corresponding antibody concentration was[Ab]_(t)=(1 mg/ml/160,000 g/mol)/44300=1.41×10⁻¹°.

n=(0.1 μg/ml)/(0.05 μg/ml)=2

Ka=(n−1)/2*(n[Ab]−[Ab] _(t))=5.75×10⁹ L/mol=1.74×10⁻¹⁰ M

TABLE 2B Anti-SAM antibodies (84-3) binding to different amount ofantigen Monoclonal anti-SAM 84-3 PLL-aza-SAM Dilution 0.05 μg/ml 0.1μg/ml 1000 3.3764 4.7244 2000 3.2469 4.5207 4000 3.1591 4.5844 80002.9176 4.3432 16000 2.6605 4.2673 32000 2.1978 3.7642 64000 1.69543.1152 128000 1.2151 2.3332

For 84-3 antibody, when coating antigen was at 0.1 μg/ml, half themaximum OD was seen at about 1:130000. The corresponding antibodyconcentration was [Ab]=(1 mg/ml/160,000 g/mol)/130000=4.807×10⁻¹¹. Whencoating antigen was at 0.05 n/ml, half the maximum OD was seen at about1:7000. The corresponding antibody concentration was [Ab]_(t)=(1mg/ml/160,000 g/mol)/7000=8.92×10⁻¹¹.

n=(0.1 μg/ml)/(0.05 n/ml)=2

Ka=(n−1)/2*(n[Ab]−[Ab] _(t))=7.29×10¹⁰ L/mol=1.37×10⁻¹¹ M

TABLE 2C Anti-SAM polyclonal antibodies binding to different amount ofantigen Polyclonal anti-SA R3 PLL-aza-SAM Dilution 0.05 μg/ml 0.1 μg/ml25 4.9225 4.9514 50 4.8524 4.9929 100 4.3849 5.1149 200 3.8518 4.8695400 3.0971 4.1802 800 2.2404 3.3979 1600 1.5083 2.4903 3200 1.03362.0391

From Table 2C, when coating antigen was at 0.1 μg/ml, half the maximumOD was seen at about 1:1500. The corresponding antibody concentrationwas [Ab]=(1 mg/ml/160,000 g/mol)/1500=4.167 10⁻⁹. When coating antigenwas at 0.05 n/ml, half the maximum OD was seen when R3 was diluted atabout 1:800. The corresponding antibody concentration was [Ab]_(t)=(1mg/ml/160,000 g/mol)/800=7.812×10⁻⁹.

n=(0.1 μg/ml)/(0.05 n/ml)=2

Ka=(n−1)/2*(n[Ab]−[Ab] _(t))=9.58×10⁸ L/mol=1.04 nM

TABLE 2D Anti-SAH antibodies (301-1) binding to different amount ofantigen Dilution from BSA-SAH BSA-SAH 2 mg/ml 1 μg/ml 0.5 μg/ml 254.5476 4.1062 50 4.775 3.5835 100 4.5725 3.2796 200 4.2861 2.9537 4004.0144 2.6701 800 3.2889 2.2375 1600 2.4796 1.7737 3200 1.6566 1.28646400 0.9619 0.783 12800 0.4866 0.4297 25600 0.2505 0.2283 51200 0.11560.1137 102400 0.0652 0.0589 204800 0.0292 0.0296

From Table 2D, when coating antigen was at 1 μg/ml, half the maximum ODwas seen at about 1:1900. The corresponding antibody concentration was[Ab]=(2 mg/ml/160,000 g/mol)/1900=6.58×10⁻⁹M, where 160,000 is themolecular weight of antibody. When coating antigen was at 0.5 μg/ml,half the maximum OD was observed at about 1:1100. The correspondingantibody concentration was [Ab]_(t)=(2 mg/ml/160,000g/mol)/1100=1.136×10⁻⁸M.

n=(1 μg/ml)/(0.5 μg/ml)=2

Ka=(n−1)/2*(n[Ab]−[Ab] _(t))=2.787×10⁸ L/mol=3.6×10⁻⁹ M

Example 5 Competitive ELISA Assay Reagents:

IB: 10 mM phosphate, 150 mM NaCl, 0.2% BSA, 0.1% Tween 20, 0.1% Proclin,pH 7.4. Samples: (a) SAM toluenesulfonate (tosylate) disulfate (Sigma)(b) SAH sodium (MW 406.39) (c) Adenosine (Sigma) (d) Methionine (Sigma).HRP-Goat-Anti-Mouse IgG (H+L) (EarthOx, San Francisco, Calif.). HRPsubstrate: one reagent substrate solution NeA-blue Tetramethyl-benzidineSubstrate. Antigen dilution buffer: IB with 0.5% BSA. Coating buffer: 50mM carbonate butter pH 9.6. Washing buffer: PBS, pH 7.5, 0.1% Tween-20.

-   -   (1). AdaM-BSA coated micro-plate was blotted, decanted and then        competitive SAM, SAH, methionine and adenosine were added in 40        μl antigen dilution butter. The 44 μl of 0.025 n/ml purified        monoclonal antibody against SAM and 16 μl of IB+Tris (100 mM)        buffer, pH 8.5 was added and together incubated at 37° C. for        1-2 hours.    -   (2). The micro-titer plate was washed three times with PBST and        blot dry.    -   (3). To each well was then added 100 μl of properly diluted        HRP-goat-anti-mouse antibody and incubated at 37° C. for 20        minutes.    -   (4). The assay mixture was then decanted, washed, and blot dry.    -   (6). To each well was added 100 of HRP substrate and incubate        for 10-15 min.    -   (7). Stop the substrate development with 50 μl/well of 2N H₂SO₄.    -   (8). OD₄₅₀ was recorded.

In order to quantify the amount of SAM in bio-samples, competitive ELISAwas developed.

The standards curve for competitive ELISA of SAM and SAH in competitiveELISA is shown in FIG. 4.

The LOGIT is defined as Ln(A/A0)(1−A/A0) where A is the OD₄₅₀ value of asample or the standard, A0 is the OD₄₅₀ value of the control well. Thenegative LOGIT value indicates that A/A0 is less than 50% and inhibitionrate (1−A/A0) is over 50%, which is an abnormal situation that shouldnot be evaluated normally.

The standard in the amount of 12.60 mg is accurately weighed and wasdissolved in small amount of DMF and then thoroughly dissolved in 0.1 mMHCl with 250 ml flask. From it, the 5 μg/ml. 2.5 μg/ml, 1.25 μg/ml,0.625 μg/ml, 0.3125 μg/ml and 0.15625 μg/ml standard solutions were madein 100 ml flasks respectively.

HRP conjugated monoclonal antibodies against SAM and SAH were generated(FIG. 5). Direct and indirect competitive ELISA methods were developedto quantitatively measure SAM and SAH.

Example 5A Competitive ELISA Assay Using Rabbit Polyclonal

Using the procedures as outlined in Example 3, competitive ELISA withanti-S-Adenosylmethionine polyclonal antibody [R3] was performed. TheAdenosylmethionine AdaM-PLL was coated into 96 wells. Serial dilution ofAdaM, S-Adenosylhomocysteine (SAH), Adenosine (Ade), L-Methionine (Met)and 1:15000 of rabbit anti-SAM serum was added. HRP conjugated Goatanti-Rabbit IgG antibody was used to develop the color.

Example 5B Competitive ELISA Using Anti-S-Adenosylmethionine MonoclonalAntibody [84-3]

The Adenosylmethionine AdaM-PLL was coated into 96 wells. Serialdilution of AdaM, S-Adenosyl-homocysteine (SAH), Adenosine, L-Methionineand 1:35000 of monoclonal antibody purified from hybridoma clone 84-3were added. HRP conjugated Goat anti-Mouse IgG antibody was used todevelop the color.

Example 5C cELISA Using Anti-S-Adenosylmethionine Monoclonal Antibody[118-6]

The AdaM-PLL was coated into 96 wells. Serial dilution of SAM standardsand HRP conjugated mouse-anti-SAM antibody clone #118-6 at dilution of1:22000 to 1:32000 was added. After incubation at 37° C. for 60 minutes,plate was washed three times and then TMB substrate was added. Afterreaction at 37° C. for 10-15 minutes, stop the reaction before measuringOD 450. FIG. 6 shows the standard curve of SAM in direct cELISA.LOGIT=Ln(A/AS0/(1−A/AS0)), where A is the OD450 value of a sample or thestandard, AS0 is OD450 value of the control well or when no antigen wasadded. The negative LOGIT value indicates that A/A0 is less than 50% andinhibition rate (1−A/A0) is over 50%.

Example 5D cELISA Using Anti-S-Adenosylhomocysteine Monoclonal Antibody[301-3]

BSA-SAH was coated into 96 wells. Serial dilution of SAH-Na standardsand HRP conjugated mouse-anti-SAH antibody clone #301-3 at dilution of1:2000 was added. After incubation at 37° C. for 60 minutes, plate waswashed three times and then TMB substrate was added. After reaction at37° C. for 10-15 minutes, stop the reaction before measuring OD450. FIG.7 shows the standard curve of SAH in direct cELISA.LOGIT=Ln(A/AS0/(1−A/AS0)), where A is the OD450 value of a sample or thestandard, AS0 is OD450 value of the control well or when no antigen wasadded. The negative LOGIT value indicates that A/A0 is less than 50% andinhibition rate (1−A/A0) is over 50%.

Example 6

Compound 1: 2′,3′-O-Isopropylideneadenosine (25 g, 82 rnrnol, 1equivalent) and dry pyridine (200 mL) were placed into a single neck,500 mL round bottom flask along with a magnetic stir bar then placedunder nitrogen atmosphere. The flask was then heated with a heat gunwhile stirring vigorously. After approximately 5 minutes all solidsdissolved. Once in solution, the mixture was cooled in an ice-water bathand stirred for 20 minutes. Tosyl-CI was added as a solid in 8 smallportions over I hour to prevent a significant exotherm. The mixture waskept at 0° C. for 5 days. Once the reaction was complete by TLC, themixture was diluted with 100 mL H₂O and 300 mL of ethyl acetate. Themixture was transferred to a separatory funnel and 100 mL of 3N HCl wasadded. The layers were separated and the organic layer was washed withfive 200 mL portions of water to remove excess pyridinium hydrochloride.The organic layer was concentrated under reduced pressure then theresidue was taken up in 100 mL of dichloromethane. This was slowly addedto a stirring solution of heptane (1.12 L) via addition funnel. Theoff-white precipitate was filtered off to give 31.1 grams of pureproduct confirmed by mass spec and IH NMR.

Example 7

Compound 2: Compound 1 (32.2 g, 70 mmol) was added to a 300 mL sealedtube along with a magnetic stirbar. Around 200 mL of a 2M solution ofmethylamine in THF was poured into the tube and the tube was sealed. Thevessel was submerged into a 50° C. oil bath then stirred for two days.The reaction vessel was taken out of the oil bath and then placed intoan ice-water bath and stirred for 30 minutes. The cap was then removedand the excess methylamine was blown out by sparging with a gentlestream of nitrogen. The residue was then transferred to a round bottomflask and concentrated under a reduced pressure. The gum-like residuewas purified by flash column chromatography (5% MeOH in DCM) to give3.51 grams oft. The structure was confirmed by I H NMR.

Example 8

Compound 3. Amine 2 (5.0 g, 15.6 mmol, 1 equivalent) was placed into asingle necked 500 mL round bottom flask. 150 mL of dry acetonitrile wasadded followed by diisopropyl-ethylamine (2.1 g, 16.38 mmol, 1.05equivalents) and stirred at 35° C. for 30 minutes. Bromobutyrate (2.55g, 14.1 mmol, 0.9 equivalents) was added drop-wise via syringe, followedby tetrabutylammonium iodide (288 mg, 0.78 mmol, 5 mol %). The mixturewas stirred at 40° C. for 5 days. The reaction mixture was thenconcentrated under reduced pressure and purified by flash chromatography(5% MeOH in DCM) to provide 5.36 grams of the desired product in 82%yield.

Example 9

Compound 4. Methylester 3 (7.76 g, 18.4 mmol, 1 equivalent) was placedin a 250 mL round bottom flask and taken up in 15 mL of methanol and 15mL H₂O and stirred for 10 minutes. Solid lithium hydroxide (1.55 g, 36.8mmol, 2 equivalents) was added and the mixture was stirred forapproximately 2 hours (until TLC and LCMS showed the completedisappearance of starting material). The crude mixture was concentratedto dryness and then carried on to the next step without furtherpurification.

Example 10

Compound 5: Approximately 7 grams of the crude lithium salt 4 wasdissolved 150 mL of 3N HCl and stirred at ambient temperature for 4hours (until starting material completely disappeared on TLC and LCMS).The crude mixture was filtered through filter paper then concentrated todryness under reduced pressure. The crude residue was purified in 5portions on a 120 gram reverse phase column eluting the product at agradient of 40% methanol in water. The purified fractions were pooledand then concentrated to dryness under high vacuum at 40° C. The productwas a brown foam that collapsed back to a brown oil upon standing. Theproduct was confirmed by HPLC. MS. and IH NMR. 4.8 grams of 99.55% pure(HPLC) product was then divided and transferred into 9 vials with a 1:1mixture of methanol and water. Each sample was then concentrated todryness under high vacuum at 40° C. until the mass remained constant.

Example 11 Blood Sample Collection Procedure

Blood samples were obtained from normal volunteers and patients withconsent. For plasma sample collection, peripheral venous blood was drawninto tubes with EDTA and mixed well. The tubes were cooled immediatelyat 4° C. and centrifuged at 2000 g for 15 minutes within 30 minutesafter blood collection to obtain plasma. The plasma was either used inmeasurement or frozen under −20° C. to −70° C. for future use. For serumsample collection, peripheral venous blood was drawn into serumseparating tubes and was placed in the refrigerator for about 2 hourstill blood coagulation was obviously visible. To help collect serumproperly, the serum tubes were centrifuged at 2000 g for 15 minutes at4° C. The serum was either used in measurement or frozen under −20° C.to −70° C. for future use.

Example 12

Blood samples were obtained from cancer patients who were hospitalizedfor chemotherapy. The samples were measured with direct competitiveELISA assay for SAM and SAH levels. The average level of SAM for cancerpatients was 103±52 nM, and the SAH level was 250±90 nM from 12 samples.The average methylation index was less than 0.5. More samples andobservations with diagnostic details, symptoms, cancer stages,progression, treatment, relapse and prognostic information are beingconducted to generate a complete profile of the human methylation indexand its relationship to various aspect of cancer at different levels.Blood samples are also collected from various patients with other typesof cancers before and after chemotherapy. The DNA methylation level willbe measured from the white blood cells as well. The relationship betweenparticular DNA methylation disorders from cancer patients andmethylation index or DNA global methylation is also expected to providefurther impact on state of health and therapeutic protocols.

Example 13

Blood samples were obtained from patients having liver disorders such ascontagious hepatitis (some accompanying bile problems includingCholestasis), liver cirrhosis, fibrosis and then analyzed for SAM andSAH levels with direct competitive ELISA. The average level of SAM inplasma was 45±8 nM from 26 samples. The level of SAM in liver disorderswas much lower than that of normal people. The SAH level was 342±129 nM,however, higher than that of normal people. The methylation index wasabout 0.13.

Example 14

Blood samples were obtained from patients having been diagnosed asdepression and then analyzed for SAM as follows:

Blood samples from depressed patients will come from The Second XiangyaMedical College Hospital Psychiatric Institute of Health for thosedepressed patients without obvious organic damages or diseases, as wellas from The Second Ningbo Hospital Neurology Department andRehabilitation Department for those depressed patients with some organicdiseases. We especially compare SAM and SAH levels in depressed patientswho take SAM-e or other medicines before and after treatment ofdepression. The level of SAM was 20±18 nM and SAH was 340±180 nM from 10samples. The methylation index was around 0.064. SAM or methylationindex can be a good indicator to personalize depression therapy and aidsin prognostic prediction. Qualitative and semi-quantitative SAM rapidtest strips are convenient choices available for patients who need todecide whether to take SAM-e or other anti-depression medicines. Thishelps direct patients to choose the medicines that best fit them.

Example 15

Patients with stage 1 cancer are examined and their SAM levels and SAHlevels are measured and the methylation indeces are calculated. Pateintswith methylation indecss of less than 2 are started with SAM once a daywhile they are undergoing chemotherapy.

Example 16

Measuring methylation index from urine (or blood sample if urine cannotbe used normally because of some special components in it or theconcentration of SAM in urine is too low) is a good way to personalizedepression therapeutics. SAMe therapeutical protocol may look like this:

For adult patients, depending on the severity of the mood and otherhealth problems, many regimens have been used, for example:Daily doses of 800-1,600 mg of SAMe by mouth for up to 6 weeks.Doses of SAMe have been given through IV or injected into the muscle,ranging from 200 to 400 mg daily at most 8 weeks.Doses of 1,000-1,600 mg have been taken by mouth daily for 15 days to 6weeks.Doses of 150-400 mg given through IV daily for 3-4 weeks are mostcommon.A dose of 400 mg of s-adenosyl-L-methionine 1,4-butanedisulphonatestable salt (Knoll Farmaceutici S.p.A., Liscate, Milan, Italy) has beeninjected into the muscle daily.Doses of 75-200 mg of SAMe have been injected into the muscle for 14-30days.Doses of 200-400 mg of SAMe per 250 milliliters of saline have beengiven through IV during the first three days of treatment, followed by400 mg of SAMe daily on days 4-14.

Use the methylation index ELISA kit developed in this invention tomeasure methylation index once every 3-5 days for patients who takehigher dosage (more than 400 mg daily) to adjust the dosages of SAMetimely. If the methylation index increases too fast (increase 5 foldsbetween measures or by 0.5 or when patients can experience obvioussymptoms associated with using SAMe.), reduce dosage is recommendedespecially for patients who have high blood pressure or othercardiovascular problems. High risk groups should have methylation indextested daily or use the SAM and SAH rapid test kid to qualitatively orsemi-quantitatively test SAM and SAH levels in blood or urine daily toensure the safety of SAM administration to avoid any side effects.

For those who take SAM-e less than 400 mg daily by mouth, havemethylation index tested, or at least SAM tested when needed, or justhave it tested weekly to get some idea of whether the dosage is rightfor the patient as well as when to stop taking the medicine.

Do not stop SAM medication unless methylation index is back to normaland stabilized for a week or two.

For those depression patients who have normal or close to normalmethylation index, do not use SAMe as the first choice of therapeutics.Instead, use other type anti-depression medicines. But methylation indexmay still be a good marker to monitor the effectiveness of othertreatment. Have methylation index tested regularly is still important todetermine when the treatment can be stopped.

Example 17

Measuring methylation index from urine (or blood sample if urine cannotbe used normally because of some specially components in it or theconcentration of SAM in urine is too low) is a good way to personalizeliver and/or cholestasis therapeutics. SAM-e therapeutical protocol maylook like this:

For adult patients, depending on the severity of the liver and/orcholestasis and other health problems, many regimens have been used, forexample:

1,600 mg of SAMe has been taken by mouth daily for 2 weeks.A dose of 1,000 mg has been injected into the vein (IV) daily for 4weeks.To treat bile flow problems in pregnancy, 500 mg of Transmetil® has beengiven by slow infusion twice daily for 14 days, followed by 500 mg ofSAMe taken by mouth twice daily until or after delivery. A dose of 600mg of Samyr® has been taken by mouth alone.A dose of 1,800 mg of Samyr® has been taken by mouth together withbeta-mimetics daily.A dose of 500 mg has been taken by mouth twice daily.Doses of SAMe that have been given include: 1,000 mg injected into themuscle daily until delivery, 200 or 800 mg given through IV daily for 20days; 800 mg given through IV daily in two divided doses until delivery;800 mg given through IV; and 800 mg given through IV over three hoursfor 20 days. A dose of 800 mg of disulfate-p-toluene sulfonate stablesalt (BioResearch, S.p.A, Milan, Italy) has been given through IV daily.

Use the methylation index ELISA kit developed in this invention tomeasure methylation index once every 3 days for patients who take higherdosage (more than 600 mg daily) to adjust the dosages of SAMe timely. Ifthe methylation index increases too fast (increase 5 folds betweenmeasures or by a certain number when patients can experience obvioussymptoms associated with using SAMe.), reduce dosage is recommendedespecially for patients who have high blood pressure or othercardiovascular problems. High risk groups should have methylation indextested daily or use the SAM and SAH rapid test kid to qualitatively orsemi-quantitatively test SAM and SAH levels in blood or urine daily toensure the safety of SAM administration to avoid any side effects.

For those who take SAM-e less than 600 mg daily by mouth, havemethylation index tested, or at least SAM tested when needed, or justhave it tested weekly to get some idea of whether the dosage is rightfor the patient as well as when to stop taking the medicine.

Do not stop SAM medication unless methylation index is back to normaland stabilized for a week or two.

Example 18 Flow Cytometry Procedure

About 1 million L-02 and Hep G2 cells were washed and fixed with 4%Paraformaldehyde for 1 hour at 4° C. Then 0.2% Triton X-100 was addedfor 15 minutes at 4° C. After washing, the cells was incubated with1:200-1:400 diluted anti-SAM monoclonal antibodies or polyclonalantibody in buffer containing goat serum at 4° C. overnight. AlexaFluor® 488 Goat Anti-Mouse IgG (H+L) antibody was added after washingwith PBS for 40 minutes at room temperature. Washed again with TBS andPBS twice before adding 1% Paraformaldehyde, then cells were run andanalyzed using Becton Dickinson flow cytometry instrument.

Use of Anti-SAM Antibodies in Flow Cytometry (FCM)

Flow Cytometry (FCM) was carried out with normal human liver cell lineL02 and hepatocyte carcinoma cell line Hep G2 cells. Two anti-SAMmonoclonal antibodies 84-3 and 118-6 as well as polyclonal antibody R3were used in the FCM assays. FIG. 11A-14 show results from two of theassays using monoclonal antibodies.

FIGS. 8A and 8B show the FCM analysis control in an assays with anti-SAMantibody. A and B showed results from two independent experiments.Normal liver cells L02 and carcinoma cells Hep G2 were stained with thebuffer without any antibody.

FIGS. 9A and 9B illustrate the FCM results from normal liver cell lineL02 and hepatocyte carcinoma cells line Hep G2 stained with anti-SAMmonoclonal antibody from clone 84-3. A and B showed results from twoindependent experiments. Average fluorescence signal in Hep G2 cells wasreduced compared to that in L02 cells, indicating SAM level is reducedduring carcinogenesis.

FIGS. 10A and 10B show the FCM results from normal liver cell line L02and hepatocyte carcinoma cell line stained with anti-SAM monoclonalantibody from clone 118-6. A and B showed results from two independentexperiments. Average fluorescence signal in Hep G2 cells was reducedcompared to that in L02 cells, indicating SAM level is reduced duringcarcinogenesis.

FIG. 11 illustrates the FCM results from normal liver cell line L02 andhepatocyte carcinoma cell line Hep G2 stained with anti-SAM polyclonalantibody R3. Average fluorescence signal in Hep G2 cells was reducedcompared to that in L02 cells, indicating SAM level is reduced duringcarcinogenesis.

FIG. 12 illustrates the FCM results from normal liver cell line L02 andhepatocyte carcinoma cell line HepG2. Cells were stained with mouseanti-SAH monoclonal antibody 301-1. Average fluorescence signal in HepG2cells was reduced compared to that in L02 cells, indicating SAH level isreduced during carcinogenesis.

After deducting the fluorescence values from the control samples,average geometric means for the 1 million cells injected, Table showedthe averages of fluorescent geometric means of 2 FCM tests. Meanwhile weobserved that the monoclonal antibody from clone 84-3 has much higherfluorescence values than the monoclonal antibody from clone 118-6 andpolyclonal antibody, suggesting there may exist some additional ordifferent properties of monoclonal antibody from clone 84-3 and theproperties do not exist in monoclonal antibody from clone 118-6 and thepolyclonal antibody.

TABLE 3 Averages of geometric means from FCM Antibody L02 HepG2 Decrease(%) Mouse anti-SAM (84-3) 164.81 48.74 70 Mouse anti-SAM (118-6) 36.0813.44 64 Rabbit anti-SAM (R3) 34.57 9.72 72 Mouse anti-SAH (301-1)103.36 56.99 45

Example 19 Immunohistochemistry Procedure

To stain cells or tissue sections, slides are blocked with blockingbuffer (1% BSA in PBS) for 30 minutes at room temperature and incubatedwith 1:10-1:20 diluted anti-SAM monoclonal antibodies or polyclonalantibodies. After rinsing twice with TBST (50 mM Tris/HCl pH 7.6, 150 mMNaCl, 0.05% Tween-20), slide was incubated with HRP labeledgoat-anti-mouse IgG for 2 hours. After twice with TBST, slides weretreated with Diaminobenzidine (DAB) reagents to visualize staining.Slides can then be counterstained with hematoxylin, dehydrated (ifrequired), and mounted for microscopy examination.

Use of Anti-SAM Antibodies in Immunohistochemistry (IHC)

Using the procedure above several normal cells and cancer cells as wellas sections from different organs were stained.

FIG. 13 shows use of anti-SAM monoclonal antibody from clone 118-6 inperforming IHC with normal and cancerous breast pathological slide. Theresults indicated dramatically reduced cytoplasmic and nuclear SAMspecific staining (brown color indicates where SAM is) in carcinomacells compared to the surrounding normal breast tissue. Cytoplasmic andnuclear areas from B showed negative or much weak or backgroundstaining. A (left): benign breast cancer adjacent to cancer region shownin B; B (right): breast cancer tissue (×400). Antibody was diluted at1:200.

FIG. 14 illustrates use of anti-SAM monoclonal antibody from clone 118-6in Performing IHC with normal and cancer lung pathological slides. Theresults indicated a dramatically reduced cytoplasmic and nuclear SAMspecific staining (brown color indicates where SAM is) in carcinomacells compared to the surrounding normal lung tissue. Cytoplasmic andnuclear areas from Picture B showed negative or background staining.Picture A (left): benign lung cancer adjacent to cancer region shown inPicture B; Picture B (right): lung cancer tissue (×400). Antibody wasdiluted at 1:200.

FIG. 15 shows the use of anti-SAM monoclonal antibody from clone 118-6in performing IHC with normal and cancerous liver pathological slides.The results indicated a dramatically reduced cytoplasmic and nuclear SAMspecific staining in carcinoma cells compared to the surrounding normalliver tissue. Cytoplasmic and nuclear areas from Picture B showednegative staining. A (left): benign liver cancer adjacent to cancerregion shown in Picture B; Picture B (right): liver cancer tissue(×400). Antibody was diluted at 1:200.

FIG. 16 illustrates the use of anti-SAM polyclonal antibody R3 was usedin performing IHC with normal and cancerous breast pathological slide.The results indicated a dramatic reduce in cytoplasmic and nuclear SAMspecific staining in carcinoma cells compared to the surrounding normalbreast tissue. Cytoplasmic and nuclear areas from Picture B showednegative staining. Picture A (left): benign breast cancer adjacent tocancer region shown in Picture B. Picture B (right): breast cancertissue (×400). Antibody was diluted at a ratio of 1:20.

FIG. 17 shows the use of anti-SAM monoclonal antibody from clone 118-6in performing IHC with normal and cancerous nephritic pathologicalslide. There were not much change in the SAM specific staining in thecarcinoma cells compared to the surrounding benign nephritic tissue.Picture A (left): benign kidney tissue adjacent to cancer region shownin Picture B. Picture B (right): kidney cancer tissue. Antibody wasdiluted at 1:200.

FIG. 18 shows the use of anti-SAH monoclonal antibody from clone 301-1in IHC with normal and cancerous breast pathological slides. The resultsindicated dramatic decrease in cytoplasmic and nuclear SAH specificstaining in carcinoma cells compared to the adjacent normal breasttissue. Cytoplasmic and nuclear areas from picture B showed reducedstaining. Picture A (left): normal breast tissue adjacent to cancerregion shown in picture B. Picture B (right): breast cancer tissue.Antibody was diluted at 1:50.

It is foreseeable that different cancer cases may give different resultsbecause each case is different, e.g. different stages, overall healthcondition, treatments used and complications of other diseases, and soon. Benign tissues stained with anti-SAM antibody were positive to adifferent degree but the malignant tissues all showed negative orreduced intracellular SAM concentration.

Immunofluorescence (IF) and IHC assays of SAM molecule of culture cells,tissue sections, biopsy cells, peripheral blood cells, and evenexfoliated cells of various tissues or origins are helpful in giving usinsights in evaluating pathological status of cells examined. High SAMlevel indicates healthy condition or benign progression or diseaseimprovement is under way. Though qualitative in nature, the differenceis so obvious that qualitative methods still serve the purpose.

On the other hand, FCM is a good way to quantify the level of SAM bycalculating the geometric means of a population of a million cells,which may provide a better statistic result than what can be seen from aslide or cell smear. The usage area of FCM is relatively narrow. It ismostly used in in vitro studies of culture cells, human peripheral bloodwhite blood cells and biopsy cells from experimental animals.

Our investigation showed the results from FCM are consistent with theresults found in IHC and IF that intracellular SAM level is drasticallyreduced in carcinoma cells whereas SAM is abundant in normal cells. Morestudies are needed to better quantify the extent to which SAM levelchanges with various situations and factors.

Example 20 Competitive Rapid Test Strip Procedure Direct Method

-   (1) Monoclonal antibodies against SAM and SAH were labeled with    colloidal gold, subsequently were sprayed evenly onto a glass fiber    mat, dried at 50° C. for 12 hours. (2) 0.2 mg/ml BSA or PLL labeled    SAM analogs or SAH (or SAH sodium salt) was evenly scribed into the    nitrocellulose membrane, dried at 50° C. for 1 hour. (3) The    absorbent paper, nitrocellulose membrane (NC membrane) from step    (2), colloidal gold mat from step (1) and sample pad (to absorb and    filter samples) were aligned and placed evenly one layer after the    other. Strips were cut into 3 mm wide with chop cutting    machines. (4) To detect samples, first test the control by inserting    the strip into 100 μl sample dilution buffer. In about 5 minutes, an    obvious purple-like band was developed. Dipped the strips into    sample solution, when the SAM or SAH levels from the samples were    higher than the predetermined cutoff values, the bands were not    shown or lightly shown, whereas if the SAM or SAH levels were lower    than cutoffs, the color bands showed up like in the control band.

Competitive Rapid Test Strip Procedure Indirect Method

-   (1) Goat anti-mouse IgG was labeled with colloidal gold,    subsequently were sprayed evenly on colloidal gold mat, dried at    50° C. for 12 hours. (2) 0.2 mg/ml BSA or PLL labeled SAM analogs or    SAH (or SAH sodium salt) was evenly evenly scribed into    nitrocellulose membrane, dried at 50° C. for 1 hour. (3) The    absorbent paper, nitrocellulose membrane (NC membrane) from step    (2), colloidal gold mat from step (1) and sample pad (to absorb and    filter samples) were aligned and placed evenly one layer after the    other. Strips were cut into 3 mm wide with chop cutting    machines. (4) Negative control was tested by inserting the strip    into 100 μl sample dilution buffer. In about 10 minutes, there was    no band developed, indicating the system behaved correctly. (5)    Positive control was tested by diluting monoclonal antibodies    against SAM and SAH properly (e.g. 5 ng/ml), dipped the strips into    it. The obvious purple-like blue bands were developed. (6) To detect    SAM or SAH from samples, mixed 50 μl monoclonal antibodies against    SAM and SAH in doubled concentration of those used in step (5) and    50 μl unknown samples, dipped the strip into it. The results were    read in about 10 minutes. If the band was clearly seen, then the SAM    or SAH levels from the samples were lower than the predetermined    cutoff values. If the bands were not shown or lightly shown, it    indicated that the SAM or SAH levels were higher than cutoff values.

Example 21 Thermal Stability of SAM and SAH

The stability of SAM and SAH at different temperatures, as well as theirstability in acid and base environment were tested. The 310 human plasmasamples were placed at room temperature for 2 hours. Compared to thesame samples that were continuously placed under 4° C., the levels ofSAM were reduced by 33.81%+19.43%, the levels of SAH were increased by21.05%±83.31%. The increased SAH was due to de-methylation of SAM in exvivi situation within a few hours of blood withdrawal. Possible reasonsfor the increased standard deviation from SAH assays might include SAHvalue varies significantly among samples, competitive ELISA assay per se(very sensitive to subtle changes) as well as variations of assay ondifferent days using different assay plates.

Additional SAM thermal stability experiments were performed with normalplasma from volunteers (our lab scientists). Volunteers' information isas follows:

TABLE 4 Normal Plasma Samples used in Stability Study IdentificationGender Age BMI General Health S1 Male 33 25.26 Very Good S2 Male 2819.60 Very Good S3 Male 55 22.04 OK S4 Female 23 20.55 Very Good S5 Male53 22.49 Very Good S6 Female 21 17.63 OK S7 Female 24 19.80 Very Good S8Mixed unknown number of plasma from blood center

Results from stability study for different temperature conditions, i.e.4° C., 15° C. (room temperature), 37° C. and 56° C., were obtainedmainly on SAM and partially on SAH measurements. Plasma from 4individual samples S3, S4, S5 and S6 were used in 4° C., 15° C. and 37°C. experiments. The results were shown in FIG. 19 A-F.

Degradation rate under 4° C. showed big variety among individuals (FIG.19 A-B). However, when samples were placed at room temperature (˜15° C.)and 37° C., SAM was degraded rapidly especially at 37° C., SAMdegradation rate reached a maximum at about 4-6 hours under 37° C. SAHlevels showed increase with time goes by, FIG. 19D showed levels ofplasma SAM and SAH change over time at 15° C. At about 6 hours afterblood withdrawal, SAM level was reduced whereas SAH level was increased.The values of SAH were recorded as >1000 nM from 6 hours to 3 days, thusSAH leaks were not shown. The level of SAH was maintained at a highlevel for about 3 days before it started to drop. SAM level went downcontinuously. It indicated that methyltransferases were still active andthe process of converting SAM into SAH may last for a couple of daysafter blood withdrawal if left under fairly cold environment. It cannotbe ruled out that inhibition of SAH converting to other metabolites ordegradation also existed causing it to stay high for quite some time. Itmeant to show the dynamics of SAM and SAH for this particular sample.Difference among samples exists. Samples S1, S2, S3 and S4 were used in56° C. experiments. The results are shown in FIG. 19F-G. SAM was quicklyundetectable in 10 minutes at 56° C., whereas SAH was dramaticallydegraded in 10 minutes and fully undetectable after about 4 hours at 56°C. SAH level was dramatically decreased within the first 10 minutes,which indicated that existing plasma SAH was first degraded, then morenewly generated SAH contributed the slight increase, and SAH level wentdown again due to the high temperature condition which caused fasterdegradation.

The chemical processes and dynamics of SAM and what forms in humanplasma are not clear. It is possible that SAM exists as both freemolecule and in association with other bio-molecules. The results ofimmunoassays reveal both free and binding forms of SAM in plasma. Thethermal stability of free and binding forms of SAM could be different,and each individual may have different portions of free orcombine/conjugated form, which partially explains why SAM from somesamples persisted relatively longer at 4° C. than that from otherparticipants (FIG. 19A). The study on the stability as measured by thedirect competitive ELISA indicated variation among participants. We donot know which form of SAM or SAH is more stable than the other form.Other possibility, e.g. certain partially degraded SAM may also be ableto bind mouse anti-SAH antibody. Further investigated is needed toclarify this.

To distinguish whether there are conjugated form of SAM and SAH in humanplasma, we used dialysis bags to dialyze plasma samples at differenttemperature for 24 hours with at least two changes of dialyzing buffer(20 mM PB, pH7.4), then measured the SAM and SAH levels before and afterdialysis.

FIGS. 20A and 20B shows that SAM and SAH were all detected at reducedlevels after dialysis compared to the controls without dialysis. Noticethat for those SAH data which were greater than or equal to 1000 nM,only 1000 nM was used in the figure. Therefore, it is obvious that theimmunoassays used in this invention can bind free SAM and SAH from humanplasma. The differences of SAM or SAH as measured before and afterdialysis (20 mM phosphate buffer, pH 7.4) for each sample representedthe free SAM or SAH molecules that escaped from the dialysis bag (MW14,000, Biosharp, USA) into the dialysis buffer, therefore they couldnot be detected from the sample collected from dialysis bag.

FIGS. 20A and 20B also show the percentages of SAM and SAH that wereleft within the dialysis bag after dialysis at 15° C. for 24 hours,which represented the amount of non-free SAM and SAH respectively. Thisobservation indicated that the non-free form of SAM was 11-25% andnon-free form (conjugated with proteins bigger than 14,000 Dalton) ofSAH was around 30%. The lines at the bottoms of FIG. 20A and FIG. 20Bshowed the minimum detection limit, whereas the values should be lessthan the minimum detection limits and should be considered trivial or noSAM and SAH left after being exposed to 37° C. for 24 hour.

As we know each individual has very different metabolite profile fromothers. Many factors including genome, neuro-endocrine, environment,health factors play roles in the outcomes of blood tests. Eachindividual has his or her unique fluid environment, for example pH,electrolyte, blood viscosity, plasma normal and abnormal components suchas drugs, etc. This may partially explain why SAM and SAH from eachindividual have different degrees of thermal stability. For example, anindividual with high pH plasma may show an increased SAM and SAHdegradation rate than another individual who has a relatively lower pHplasma environment.

Example 22 Stability of SAM and SAH at Different pH Values

The stability of SAM and SAH was also tested in acidic and basicconditions at 15° C. Normal mixed plasma sample (S8) (with pH 7.84),samples S1, S2, S3, S4 (pH 7.0-7.5) were used in the experiments. 10 μl5M HCl was added to 1260 μl plasma to adjust its pH to 5.5-6.0. Inanother set of samples, 2 μl 5M NaOH was added to 1260 μl plasma toadjust its pH to 8.0-8.5. Before using these acidic or basic samples toconduct the immunoassay, 1.1 μl 5M NaOH was added to 180 μl acidicsamples; 0.376 μl 5M HCl was added to 180 μl basic samples to make themneutral. Meanwhile, standards were also added the same amount of acidand base as the samples. All samples have the same trend, FIGS. 21A and21B only showed the dynamic SAM and SAH with pH from one sample S2.

In FIG. 21A, SAM shows degradation rate increased with pH values. Thisobservation was consistent with a prior publication regarding pHstability of SAM (Parks, L W, et al. jbc.org Aug. 23, 1957). Itindicated that acidic environment slowed down SAM degradation. FIG. 21Bindicated both acidic and basic environment speeded up plasma SAHdegradation. However basic environment has a stronger impact than acidicenvironment. The SAH data at 1 day time point, the SAH levels for pH7.0-7.5 and pH 5.5-6.0 curves were measured to be greater than 1000 nM.The experiment has maximum detection limit as 1000 nM, only 1000 nMpoints were plotted, therefore the spikes at 1 day time point could notbeen see for pH 7.0-7.5 and pH 5.5-6.0 curves. This figure is meant toshow the dynamics of SAH level for this particular sample. Differenceamong samples exists.

Example 23 Healthy Human Plasma SAM and MI as a Function of Gender andAge Study #2

Normal samples were strictly evaluated through series of examination andproven disease free. Normal human serum was collected and frozen within2 hours with serum separator tubes or SSTs. Serum samples were storedfrozen at −80° C. The samples were analyzed according to theimmunoassays of the present invention.

TABLE 5 SAM Level and Methylation Index (MI) in Normal Subjects % ofSAM > % of SAM > % of % of % of Case # 240 nM 120 nM Avg. ± Stdev MI > 2MI > 1 MI < 0.5   Male (41) 56.09 90.24 340.51 ± 211.70 43.90 43.4112.20 Female (40) 82.50 100 433.96 ± 213.02 52.50 72.50 5.00  Total (81)69.14 95.06 386.66 ± 216.20 46.91 67.90 8.64

Table 5 shows that about 56.09% of males whose SAM levels were higherthan 240 nM whereas 82.50% of females whose SAM levels were higher than240 nM. Methylation Index (ratio of SAM and its de-methyl productS-adenosylhomocysteine, SAH) was also higher in females than in males.When methylation index was less than 0.5, it is considered as sub-healthor disease condition. There are only 5% of female whose MI was less than0.5 whereas there were 12.20% of males whose MI was less than 0.5. It isbelieved that SAM is a health indicator: the higher the SAM, the betterthe health status one could be at. SAH is just the opposite, the higherthe SAH, the worse the health status one could be. Normal females havehigher SAM level and methylation index than normal male individuals. Howdoes this relate to the fact that females have a relatively longerlifespan and better disease-resistance capability, which remains to befurther investigated.

Before performing any statistical analysis, we first examined whetherdata sets in our studies fit normal distribution. We used R to test thedata distribution property and found the means and standard deviationsof SAM, SAH and MI for healthy and diseased samples all fit normaldistribution (FIG. 22A-22B).

FIG. 23 shows the average of SAM levels by age groups and genders.Statistical analysis with R was performed by Sydney Wong (We sincerelyappreciate her contribution to the analysis for study #2)

TABLE 6A ANOVA Results for Different Age and Gender Response ExplanatoryDataset Variable Variable p-value Significance Healthy SAM Age Group0.03437 ** Healthy SAH Age Group 0.4964 Healthy MI Age Group 0.1058Healthy SAM Gender 0.05115 * Healthy SAH Gender 0.584 Healthy MI Gender0.8752 Note: * represents significance at significance level α = 0.1. **represents significance at significance level α = 0.05. *** representssignificance at significance level α = 0.01. **** representssignificance at significance level α = 0.001.

Based on the ANOVA table, we see that Age Group is a significant factorwith respect to SAM levels in healthy patients. So we will do pairwiset-tests for each pair of levels of Age Group.

TABLE 6B ANOVA Results for Pairwise Age Comparisons Age Group 1 AgeGroup 2 p-value Significance 20-30 31-40 0.01728

20-30 41-50 0.03793

20-30 51-80 0.00568

31-40 41-50 0.7461 31-40 51-80 0.7815 41-50 51-80 0.9081So clearly, by the pairwise t-test results, the SAM levels for thehealthy patients aged 20-30 are statistically significant from those ofthe other age groups. And, looking at the plot, of mean SAM levels ofhealthy patients by Age Group, we see that the SAM levels of healthypatients aged 20-30 are significantly higher than those of the other agegroups.

After doing a pairwise t-test for SAM levels of healthy patients bygender, we get a p-value of 0.05117 which is statistically significantat an α=0.1 level. The mean of the female SAM levels is 433.96 and themean of the male SAM level is 340.51, which means the SAM levels forhealthy females are significantly higher than those of healthy males.

Example 24 SAM and MI in Blood Center Plasma and Diseased Plasma Study#3

310 cases of normal plasma samples were frozen at −20° C. within 3-10hours with EDTA as anticoagulants and analyzed using the immunoassays ofthe present invention. The study was done with normal samples afterexcluding existence of infectious diseases such as virus hepatitis, HIVinfection, Syphilis, Gonorrhea from a blood center with blood donatorsbetween 18-60 years old.

TABLE 7A Disease Case Information in Study #3 Cerebrovascular diseases +Parkinson's Disease 45 + 3 Diabetes 43 HBP 22 Heart diseases 51Inflammation 35 Kidney disease + Diabetes Kidney disease 24 + 2 Liverdiseases 30 Pulmonary diseases 36

TABLE 7B Cancer Case Information in Study #3 Bladder Cancer 2 BreastCancer 3 Colon Cancer 16 Esophagus Cancer 3 Gallbladder Cancer 1 Lipoma2 Liver Cancer 23 Lung Cancer 75 Lymphoma 2 Multiple Myeloma 1 OvaryCancer 2 Prostate Cancer 5 Throat Cancer 4 Thymoma 1 Thyroid cancer 1Uterus Cancer 5 Vascular Cancer 1 Cancer 27

TABLE 7C Brain Disease Information in Study Cerebrovascular diseases 20Depression 10 Parkinson's Disease 10

TABLE 8 Distribution of Normal Human Plasma SAM Levels in Females andMales Male Female SAM (nM) No. % No. %  <30 0 0 1 1.01 30-60 9 4.27 33.03  60-120 44 20.85 9 9.09 120-240 70 33.18 33 33.33 240-480 75 35.5539 39.39 480-960 13 6.16 14 14.14 >960 0 0 0 0 Avg. SAM 211 232.86 99296.92 Stdev. SAM 211 149.56 99 185.12

TABLE 9 Distribution of Normal Human Plasma SAM Levels in Different AgeGroups Age 18-30 Age 31-40 Age 41-50 Age 51-60 SAM (nM) No. % No. % No.% No. %  <30 1 0.73 0 0 0 0 0 0. 30-60 1 0.73 5 6.10 4 5.33 2 12.50 60-120 17 12.41 16 19.51 17 22.67 3 18.75 120-240 42 30.66 28 34.15 3040.00 3 18.75 240-480 58 42.34 28 34.15 20 26.67 8 50.00 480-960 1813.14 5 6.10 4 5.33 0 0 >960 0 0 0 0 0 0 0 0  <60 2 1.46 5 6.10 4 5.33 212.5 >240 76 55.47 33 40.25 24 32 8 50 Total No. 137 82 75 16

TABLE 10 Normal Human Plasma SAM Levels in Age Groups and Genders AgeGroup Gender Average Standard deviation Age 18-30 Male 255.55 144.44Female 360.39 206.59 All 290.75 174.34 Age 31-40 Male 237.91 173.28Female 256.46 148.63 All 242.66 166.62 Age 41-50 Male 196.30 126.92Female 234.11 156.75 All 208.40 137.24 Age 51-60 Male 169.42 95.86Female 226.61 104.93 All 198.01 101.48

FIG. 24 also shows that on the average women had higher SAM level(296.92 nM) than men (232.86 nM) (see Table 8), which was about 28%higher. This was hardly because of the diet factor as we believed menand women had the same diet in the same culture, same geographicallocation and with the same social status. There must be some fundamentalreasons that warranted the consistent difference observed here. This waslikely related to the metabolism and genetic reasons, which remained tobe further discovered.

SAM levels in female were higher than in males, which was consistentwith previous Study #2. FIG. 24 showed the average SAM levels indifferent age groups and gender groups. SAM levels continuously goingdown as ages go up, men or women. This indicated SAM might be involvedin aging process. From Table 8, we could see higher percentage of peopleolder than 51 years old have SAM level less than 60 nM, indicating aspeople get older, SAM level is decreased. Table 10 showed as the ageincreased, the percentage of people who had decreased SAM levelincreased.

Patient samples were collected from clinical labs with no furtherinformation about disease condition, status and treatment. Samples werehandled similarly to the samples described in Study #3. Statisticalanalysis with R was performed by Dr. Huaitian Liu (We sincerelyappreciate his contribution to the analysis for Examples 24, 25 and 26)

Table 11 shows t-test results by comparing 81 cases of normal case fromExample 23 with the diseases in Table 7A, which included kidney diseases(Renal cysts, kidney stones, hydronephrosis, cancer), liver diseases(hepatitis, cirrhosis) and diabetes were more significant than pulmonarydiseases (chronic obstructive pulmonary emphysema, cancer, pneumonia,TB), cardiovascular & cerebrovascular diseases (coronary atheroscleroticheart disease, hypertension, Cerebrovascular diseases, embolism, lowerextremity atherosclerosis obliterans), inflammation (gastrointestinaldiseases, ulcer, intestinal obstruction, asthma, ureteral stones,prostate benign, ulterus benign, nasopharyngeal and breast diseases,blood system disorders, hernia, etc.).

TABLE 11 Results of t-test in Diseases from Table 7A Response VariableMean p-value Significance SAM(Cerebrovascular 357.9406 0.4365 diseases)SAH(Cerebrovascular 353.175  0.001237 *** diseases) MI(Cerebrovascular1.148035 2.31E−05 **** diseases) SAM(Diabetes) 262.3474  0.001136 ***SAH(Diabetes) 372.0686  0.003831 *** MI(Diabetes) 0.8619158 9.20E−08**** SAM(HBP) 288.3645 0.0389 ** SAH(HBP) 358.1118  0.01378 ** MI(HBP)0.9178064 3.93E−07 **** SAM(Heart diseases) 315.962  0.07938 **SAH(Heart diseases) 440.9451 5.95E−07 **** MI(Heart diseases) 0.78227015.61E−09 **** SAM(Inflammation) 223.732 3.05E−06 **** SAH(Inflammation)292.012 0.2051 MI(Inflammation) 0.8193137 1.91E−08 **** SAM(Kidneydisease) 307.6073 0.1077 SAH(Kidney disease) 497.1204 7.76E−06 ****MI(Kidney disease) 0.6976807 3.69E−09 **** SAM(Liver diseases) 356.34070.5037 SAH(Liver diseases) 444.0027 1.22E−06 **** MI(Liver diseases)0.8921861 2.72E−07 **** SAM(Pulmonary diseases) 393.3392 0.8827SAH(Pulmonary diseases) 486.5144 4.24E−08 **** MI(Pulmonary diseases)0.7916811 5.27E−09 ****

As can be seen from Table 11, in all diseases, MI was significantlyreduced. In the case of inflammation, SAM was significantly reduced andSAH change was not significant. In cerebrovascular, liver, kidney andpulmonary disease, SAM changes were not significant but SAH wassignificantly increased.

TABLE 12A Results of t-test in Cancers from Table 7B Response VariableMean p-value Significance SAM(Liver Cancer) 278.0652 0.03451 **SAH(Liver Cancer) 293.35 0.3288 MI(Liver Cancer) 1.562109 0.1131SAM(Lung Cancer) 262.5039 0.001248 *** SAH(Lung Cancer) 326.98770.007807 *** MI(Lung Cancer) 0.9710702 3.67E−06 **** SAM(Other Cancer)283.3233 0.00116 *** SAH(Other Cancer) 394.7301 5.61E−06 **** MI(OtherCancer) 0.8156412 8.12E−09 ****

We regrouped cancer cases from Table 7B into three cancer groups (livercancer 23 samples, lung cancer 75 samples, other cancers 76 samples). Ascan be seen from Table 12, except for liver cancer samples, the resultsfrom other cancer samples indicated that SAM and MI was significantlyreduced and SAH was significantly increased. As we had no furtherinformation about the 23 cancer patients, it is very likely that thepatients were given SAM treatment as it is a routine to supplement SAMpills to patients with liver disorders upon hospitalized. Otherwise, theSAM levels could be lower. Therefore, MI would be significantly loweredas in the case of other cancers.

TABLE 12B Results of ANOVA in Cancers from Table 7B Response ExplanatoryDataset Variable Variable p-value Significance Liver Cancer SAM AgeGroup 0.4718 Liver Cancer SAH Age Group 0.0974 * Liver Cancer MI AgeGroup 0.007917 *** Liver Cancer SAM Gender NA (all NA males) LiverCancer SAH Gender NA NA Liver Cancer MI Gender NA NA Lung Cancer SAM AgeGroup 0.009508 *** Lung Cancer SAH Age Group 0.005919 *** Lung Cancer MIAge Group 0.08754 * Lung Cancer SAM Gender 0.07647 * Lung Cancer SAHGender 0.6086 Lung Cancer MI Gender 0.0001109 **** Other Cancer SAM AgeGroup 0.06088 * Other Cancer SAH Age Group 0.05491 * Other Cancer MI AgeGroup 0.5193 Other Cancer SAM Gender 0.2516 Other Cancer SAH Gender0.8028 Other Cancer MI Gender 0.4848

Table 12B showed the results of ANOVA for cancer samples described inTable 7B. It indicated that age and gender groups were significantfactors with respect to MI and SAM (for SAH, only age group not gendergroup was significant) in lung cancer cases. With liver cancer cases, asonly male patients were observed, therefore no result was obtained ongender group but age factor is significant with respect to MI. In othercancers, age group factor is slightly significant at significant levelα=0.1 with respect to SAM and SAH, not MI.

TABLE 13 Results of t-test in 80 Lung Cancer Samples Response VariableMean p-value Significance SAM 137.6983 9.60E−16 **** SAH 462.5221.03E−10 **** MI 0.3840688 1.08E−12 ****

As can be seen from Table 13, 80 cases of lung cancer samples weretested and analyzed with R as well. The results indicated that SAM andMI were significantly reduced and SAH was significantly increased incancer patients compared with normal people.

TABLE 14 Results of t-test in Diseases from Table 7C Response Signif-Diseases Variable Mean p-value icance Cerebrovascular diseases SAM415.4277 0.6064  Cerebrovascular diseases SAH 363.3671 0.03015 **Cerebrovascular diseases MI 1.163449 2.03E−05 **** Depression SAM337.385 0.3531  Depression SAH 442.379 0.01176 ** Depression MI 0.870514.81E−06 **** Parkinson's Disease SAM 285.5726 0.07684 * Parkinson'sDisease SAH 794.5792 0.06972 * Parkinson's Disease MI 0.74988 7.18E−06**** Depression + SAM 363.4532 0.5513  Parkinson's Depression + SAH490.9231 0.00325 *** Parkinson's Depression + MI 0.9868218 4.95E−07 ****Parkinson's

As can be seen from Table 14, MI is a better and sensitive indicator foridentifying diseases such as cerebrovascular disorders, depression andParkinson's disease, and it was significantly reduced in these diseases.

TABLE 15 SAM Levels and Methylation Index (MI) in Brian Diseases % of %of % of % of % of SAM > SAM > MI > MI > MI < Diseases (Case #) 240 nM120 nM 2 1 0.5 Normal (311) 90.36 96.79 6.95 42.47 16.98 Normal (81)69.14 95.06 46.91 77.90 8.64 Cerebral hemorrhage (10), 85 90 0 65 0embolism (6), infarction (4) Parkinson's Disease (10), 70 90 0 25 20Depression (10)

As can be seen from Table 15, the percentages of SAM levels greater than120 nM were comparable between normal people and patients withParkinson's disease or depression, However, significant increase in thepercentage of patients having MI less than 0.5 (about 8.64% normalpeople with MI<0.5 from Table 7 vs. 20% of Parkinson's disease anddepressed patients), and significant decrease in the percentage ofpeople having MI greater than 1 were observed, It suggested that MIcould be a good marker for Parkinson's disease and depression.

The less the MI, the higher probability Parkinson's disease ordepression might occur. On the other hand, in the cases ofCerebrovascular diseases, such as cerebral hemorrhage, embolism andinfarction, no obvious changes in SAM levels and MI were observed inthis study.

Table 15 summarizes the ranges and average of MI for cases in Example24. It is consistent with the t-test results that normal people hassignificantly higher MI (averagely 2.2) than diseased patients(averagely <1.56). The obvious difference between normal and diseasedgroup can also been seen from the maximum MI.

In the cases of cancers, we can see some samples had MI values fellbetween 4 and 6, therefore the average MI in cancer groups wererelatively higher than other diseased groups. The relatively higher MIin cancer samples is due to relatively higher SAM. This is consistentwith a report by Alissa K. et al (Chest. 2007. 132(4): 1247-1252.) whichserum SAM levels were elevated in patients with lung cancer as comparedto smokers with benign lung disorders and healthy nonsmokers. There wereno significant correlations between SAM levels and tumor cell types,nodule size, or other demographic variables. The temporarily high SAMlevels may be caused by release of intracellular SAM from cells intoblood stream (as can be confirmed by IHC staining of SAM from cancercells) at some stage of cancer progression. Therefore, in our cancersamples, we could see wide ranges of MI values observed in all types ofcancers. As only certain stage of cancer development that SAM release issignificant, once release is completed, SAM level in blood stream won'tmaintain at the high level, which contributes to the big ranges of SAMlevels observed for all cancer types.

In the case of 68 cases of cerebrovascular diseases, there is only onesample has MI as 5.67. All others were less than 2.09. If we considerthat sample as abnormal (or an outlier), the average MI for that groupwould be less than 1. Therefore, cerebrovascular diseases would be amongother diseases in evaluating the range and average value of MI.

TABLE 16 Range and Average of MI from Study Group Range of MI Average MINormal 0.40-6.50 2.22 All diseases 0.07-5.67 0.87 Cerebrovasculardiseases 0.36-5.67 1.06 Parkinson's Disease 0.08-1.86 0.75 Depression0.36-1.94 0.87 Diabetes 0.11-3.86 0.86 HBP 0.10-1.19 0.92 Heart disease0.07-1.95 0.78 Inflammation 0.28-2.13 0.82 Kidney disease 0.10-1.92 0.7Liver diseases 0.22-2.50 0.89 Pulmonary diseases 0.13-1.49 0.79 OtherCancers 0.10-5.42 0.81 Liver Cancer 0.13-4.98 1.56 Lung Cancer 0.06-5.420.68

Example 25 Samples from a Group Annual Health Examination Study #4

The study was done among a random group of people (a manufacture inChangsha, Hunan Province, China) from the test results of its annualphysical examinations. Normal plasma was processed and frozen within 5-7hours after blood withdraw with EDTA as an anticoagulant. Plasma wascollected and stored frozen below −20° C. Subsequent examinations andother tests reviewed by doctors before diagnosis were finalized and thengroup the cases accordingly. The case information is summarized in Table17. We performed the t-tests on the 303 healthy individuals and 271patients including sub-healthy group people. The results were shown inTable 18, which indicated that diseased group has significantly low SAMand methylation index as well as significantly increased SAH levelscompared to healthy people. This study was in agreement with Study #3that methylation index is a healthy indicator to distinguish healthysubjects from un-healthy subjects. Methylation index can be used as ascreening biomarker to identify the existence of any of potentialunfavorable health conditions of human beings though furtherexaminations are necessary for a diagnosis of any potential diseases. Itis therefore recommended as a part of annual health examination panel.

TABLE 17 Sample Types and Information in Annual Health Study Case TypeCase # Healthy People 303 Sub-health condition: at least one blood testswere off-normal 53 but no clear diagnosis was given Benign liverdisease: Fatty liver, hepatic cysts, schistosoma 76 liver, hepatichemangioma, diffuse parenchymal liver disease, alcoholic liverHypertension (HBP) 41 Kidney disease: Renal cysts, kidney stones,hydronephrosis 15 Cardiovascular diseases: heart attack,atherosclerosis, high 29 blood lipid Inflammation: gall bladderGallbladder polyps, gallbladder 16 stones, cholecystitis, TB, breastslobular, gynecological Diabetes 19 Other diseases: cerebrovascularcomplications, prostatic 22 hypertrophy, pulmonary diseases

TABLE 18 Results of t-test for Healthy vs. all other Disease andSub-healthy Groups Study Response Variable p-value Significance SAM0.0001353 **** SAH 0.01636 ** MI 0.08857 *

Example 26 SAM and MI in Liver Diseases Study #5

The study was done for patients with variety of liver diseases from thein-patient department of Infectious Diseases in the First AffiliatedHospital, Xiangyang Medical School, Zhongnan University, China. Sevencancer samples were obtained from patients with definite diagnosis fromthe Out-patient Department. The EDTA plasma samples were collected andfrozen in −20° C. within 30 minutes after blood drawn.

Our experiment showed no differences in SAM and SAH levels using serumor plasma samples. Therefore, we employed the data from normal samplesin Table 6 to compare with the data from liver diseases in this study.Using direct competitive ELISA, we tested 46 hepatitis samples (severevirus hepatitis), 14 liver cancer samples, 20 various degrees ofcirrhosis plasma and 19 advanced stage of cirrhosis when liver functioncannot be sustained (liver failure). Table 19 showed differences of SAMlevels and MIs in different groups. The SAM levels and MIs were muchreduced in all liver disease types. None of the liver disease samplestested had MI greater than 2 and very few of liver carcinoma andhepatitis had MI greater than 1. In the case of liver failure, SAM levelwere all below 120 nM. It is not hard to understand why this may happenas liver is the main organ in human body to produce SAM and SAH. Themore destructive the liver disease, the less SAM and SAH are to beproduced.

TABLE 19 SAM Levels and Methylation Index (MI) in Liver Diseases # SAM ># SAM > % of % of % of Group # Case 240 nM(%) 120 nM(%) MI > 2 MI > 1 MI< 0.5 Normal 81  56(69.14%) 77(95.06%)  46.91 67.90 8.64 Hepatitis 46 2(4.35%) 9(19.57%) 0 2.56 94.87 Carcinoma 14 1 (7.14%) 4(28.57%) 0 7.1485.71 Cirrhosis 20 1 (5.00%) 3(15.00%) 0 0 89.47 Liver failure 19 0 0 00 100

As it is well known that SAM and SAH levels can be affected by gender,age, race, diet, body weight, neuro-endocrine, health and other unknownfactors and are part of normal metabolites, about 10-80% of the standarddeviation may be observed depending on situations. When normal plasmaaverage SAM and standard deviation was at 386.66±216.20 nM, hepatitis atmuch reduced 101.42±83.12 nM, liver carcinoma at 104.96±82.63 nM,cirrhosis at 92.95±62.41 nM, liver failure at 66.46±29.77 nM. Furtheranalyses using 120 nM and 240 nM as cutoff values separately to identifyliver diseases with SAM level, the detected numbers and detection ratesfor all groups were calculated (See Table 20).

TABLE 20 SAM Levels in Diagnosis of Liver Diseases SAM (nM) SAM = 120 nMCutoff SAM = 240 nM Cutoff Case Standard Detected Detection DetectedDetection Group No. Average Deviation Number Rate (%) Number Rate (%)Normal 81 386.66 216.20 2 2.46 20 24.69 Hepatitis 46 101.42 83.12 3780.43 44 95.65 Carcinoma 14 104.96 82.63 10 71.43 13 92.86 Cirrhosis 2092.95 62.41 17 85.00 19 95.00 Liver failure 19 66.46 29.77 19 100 19 100

Using the 240 nM SAM cutoff, detection rate for normal group (falsepositive) was 24.69%, whereas detection rates for all liver diseasegroups were 92.86-100% (false negative rates were reduced to 0-7.14%).With the 120 nM SAM cutoff, detection rate for normal group (falsepositive rate) was reduced significantly to 2.67% whereas detectionrates for liver diseases were still as high as 71.43-100% ((falsenegative rates were between 0-28.57%). With higher cutoff, falsepositive rate can be as high as 24.69%. By reducing the cutoff, falsepositive rate was much decreased, yet detection rates for liver diseasegroups were also decreased causing the false negative rate to increase.For liver failure group, the detection rate was still 100% with 120 nMcutoff or 240 nM cutoff Refer to other liver biomarkers from lab resultsor other relevant examination reports when SAM level falls within120-240 nM or even if below 120 nM. The best way to the unavoidableoverlap of values of a biomarker between diseased and healthy conditionsis to get to know each individual's baseline reference profile anddynamically monitor the changes.

Example 27 SAM and MI in Healthy Human Saliva STUDY #6

No reports on measuring SAM and SAH levels from human saliva samples hasbeen reported in the prior art. In this study, we tested 30 normal humansaliva samples from volunteers. Samples (a) were collected about 1-2hours post-breakfast after mouth-rinse a couple of times; samples (b)were collected right after samples (a) and after brushing teeth withtoothpaste; samples (c) were collected 2-4 hours after samples (b) werecollected without any food intake in between. The samples werecentrifuged at 4° C. for 5-10 minutes at 10,280×g and frozen till thenext day for measurement of SAM and SAH of different time points at thesame time with and without dilution. The SAM and SAH competitive ELISAkits were used to measure SAM and SAH in phosphate incubation buffer aswell as plasma matrix, respectively.

The results indicated that after brushing teeth with toothpaste, SAM andSAH levels were shown increased significantly, which is believed to benon-specific inhibition causing OD450 to be significantly reduced,thereby concentrations of this group were calculated much higher. Therewere a few samples that also showed non-specific inhibition includingvolunteers who were taking traditional Chinese medicines and smokedheavily around the time of saliva collection. These samples were removedfrom the results.

An ELISA kit designed for measuring plasma samples was used formeasuring saliva samples and the results are shown in Table 21. Theresults showed the nonspecific increase caused by brushing teeth on SAMis 7.4-8.4 folds, and on SAH is 2.9-3.5 folds. The SAM values fromsample (c) (i.e. 2-4 hours after brushing the teeth without any foodintake) is about 2.8-3.1 folds higher than samples (a) (samples taken1-2 hours after breakfast and before brushing teeth), and SAH about1.8-2.6 folds higher. The ranges are also very broad. The reason why SAMand SAH values from samples (c) were higher than those of sample (a) maybe related to oral environment especially human mouth flora, which isconsidered to be determined by each person's genetic background ormakeup. Genetic factors have a lot much impact on each individual'smouth flora than external environmental factors. Like seen from theresults of plasma samples, the broad ranges and big standard deviationsof SAM and SAH values from saliva samples also indicated that SAM andSAH levels are affected by plenty of enzymes and environmental moleculesand factors in several metabolic pathways, many of which are determinedby differenced of genetic materials of each individual. Thus, a varietyexists among individuals. In order to best monitor SAM and SAH levelsfrom saliva, keep oral environment (food, medication, smoking, drinks,alcohol, etc.) similar between tests and collect saliva samples at afixed time of a day.

TABLE 21 SAM and SAH levels from saliva samples Mean Standard Samples(nM) deviation (nM) Range SAM Avg. Sample (a) 102 71 (30 nM, 257 nM)Avg. Sample (b) 852 719  (30 nM, 2449 nM) Avg. Sample (c) 309 241 (30nM, 664 nM) SAH Avg. Sample (a) 184 119 (31 nM, 717 nM) Avg. Sample (b)612 361 (122 nM, 1112 nM) Avg. Sample (c) 479 322 (39 nM, 695 nM)

All patents, patent applications and publications cited in thisapplication including all cited references in those patents,applications and publications, are hereby incorporated by reference intheir entirety for all purposes to the same extent as if each individualpatent, patent application or publication were so individually denoted.

While the many embodiments of the invention have been disclosed aboveand include presently preferred embodiments, many other embodiments andvariations are possible within the scope of the present disclosure andin the appended claims that follow. Accordingly, the details of thepreferred embodiments and examples provided are not to be construed aslimiting.

It is to be understood that the terms used herein are merely descriptiverather than limiting and that various changes, numerous equivalents maybe made without departing from the spirit or scope of the claimedinvention.

What is claimed is:
 1. A method of assessing one or more health statusesof a subject, the method comprising: determining the methylation indexstatus in a test sample from the subject; said methylation index beingcalculated by determining the levels of SAM and SAH using anyimmunoassays; comparing one or more of the determined methylation indexstates to one or more baseline reference methylation index states;wherein a difference, lack of a difference, or both in one or more ofthe determined methylation index states and one or more of the baselinereference methylation index states indicates one or more statuses ofhealth of the subject.
 2. The method of claim 1 wherein said immunoassayis an ELISA or other homogeneous immunoassays.
 3. The method of claim 1,wherein said immunoassay is performed in a strip or other dryquantitative or qualitative detection devices.
 4. The method of claim 1,wherein said health status refers to being healthy mentally andphysically.
 5. The method of claim 1, wherein said health status iseffectiveness of medical treatment.
 6. The method of claim 1, whereinsaid health status is longevity status.
 7. A method for screening,diagnosing and monitoring a disease or condition in a subject comprisingthe steps of: (a) obtaining a biological sample from a patient to bescreened, diagnosed or monitored; (b) determining using an immunoassaythe quantity of SAM and SAH in said biological sample; (c) calculatingthe methylation index of said biological sample; (d) comparing saidmethylation index of step (c) to the patient's baseline referencemethylation index profile; and (e) determining if the quantity(ies) ofsaid SAM, SAH and methylation index in said biological sample is(are)indicative of the presence, absence or stages of any diseases orconditions.
 8. The method of claim 7, wherein said reference profile forscreening, diagnosing and monitoring, comprises levels of SAM, SAH andmethylation index that are differentially present at a level that isstatistically significant, the profile profiling being of at least oneof one or more disease, the at least one reference methylation indexprofile profiling at least one of: one or more disease, cerebrovasculardiseases, Parkinson's disease, depression, diabetes, HBP, heart disease,inflammation, kidney disease, liver diseases, pulmonary diseases, lungcancer, liver cancer and other cancers.
 9. A method of claim 7, forscreening and diagnosing whether a subject suffers from liver disease,comprising: identifying, using an anti-SAM antibody, whether saidsubject has a serum or plasma human SAM concentration at a giventhreshold value, and thereby diagnosing whether the subject suffers fromliver disease.
 10. The method of claim 9 wherein said liver disease isacute liver disease selected from the group consisting of liver failure,acute viral hepatitis, carcinoma and cirrhosis.
 11. The method of claim9 wherein said liver disease is chronic liver disease, selected from thegroup consisting of fatty liver, hepatic cysts, schistosoma liver,hepatic hemangioma, diffuse parenchymal liver disease, and alcoholicliver.
 12. The method of claim 9, for monitoring liver health in apatient, further including measuring the level of one or more additionalbiomarkers selected from the group consisting of bilirubin (total orfractionated, conjugated or unconjugated), ammonia,carbohydrate-deficient transferring (CDT), alanine aminotransferase(ALT), alkaline phosphatase (ALP), serum glutamic pyruvic transaminase(SGPT), aspartate aminotransferase (AST), serum glutamic oxaloacetictransaminase (SGOT), albumin, total protein (i.e., plasma proteins),gamma-glutamyl transferase (GGT), gamma-glutamyl transpeptidase (GGTP),lactic acid dehydrogenase (LDH), prothrombin time, or combinationsthereof and comparing said SAM levels and said biomarkers in said sampleto a level of said SAM and said biomarkers of to a normal controlsample; and determining the health condition of the liver in saidpatient based on said comparison.
 13. The method of claim 7 wherein saidimmunoassay is an ELISA or other homogeneous immunoassays.
 14. Themethod of claim 7, wherein said immunoassay is performed in a colloidalgold strip or other dry quantitative or qualitative detection devices.15. A method of use comprising: (a) obtaining at least one sample from asubject; (b) determining the level of SAM, SAH and methylation index asprognostic predicting markers in the samples; (c) treating a subjectwith a potential therapeutic intervention based on the level ofprognostic predicting markers in the subject; (d) determining the effectof the therapeutic intervention based on the level of a prognosticpredicting marker or markers; and (e) using the marker response topredict the effectiveness and prognosis of the intervention.
 16. Themethod of claim 15, for determining the prognosis of acute and chronicliver disease in a subject, the method further comprising analyzing thesample using an immunoassay for the level of SAM; and then correlatingthe level of SAM in the sample to the prognosis of said acute andchronic liver disease in the subject.
 17. The method of claim 16 whereinsaid acute liver disease is selected from the group consisting of liverfailure, hepatitis, carcinoma, cirrhosis, fatty liver, alcoholic liver,hepatic cysts, schistosoma liver, hepatic hemangioma, diffuseparenchymal liver disease.
 18. The method of claim 15, wherein thetherapeutic intervention comprises cardiac therapy, cancer therapy,autoimmune therapy, hepatic treatment, kidney therapy, metabolicdisturbance therapy, anti-bacterial therapy, anti-viral therapy,anti-fungal therapy, eye therapy, infertility therapy, neurodegenerativetherapy, mood problem therapy or combination thereof.
 19. A method fordetermining whether an immunocompromised patient is susceptible to PCPinfection which method comprises: (a) obtaining a biological sample froma patient; (b) determining using an immunoassay the quantity of SAM insaid biological sample; (c) comparing the quantity of SAM in saidbiological sample to the patient's baseline reference; and (d)determining if the quantity of said SAM in said biological sample isindicative of the presence, absence or status of the PCP infection. 20.The method of claim 19, wherein said immunocompromised patient has HIV,is an organ transplant recipient, has severe nutritional disorder, saidpatient is undergoing chemotherapy/radiotherapies and said patient hasadvanced cancer.